Estimating the contributions of mobile sources of PAH to urban air using real-time PAH monitoring.

Motor vehicles are a significant source of airborne polycyclic aromatic hydrocarbons (PAH) in many urban areas. Traditional approaches used in determining the relative contributions of individual vehicle types to the total amount of PAH in air have been based on the analysis of integrated samples of airborne particles and gases for the presence of chemical tracers indicative of the vehicles from which the chemicals derived. As an alternative, we have used a photoelectric aerosol sensor (PAS) capable of measuring PAH levels in real-time in the emissions plumes from motor vehicles. We placed the PAS near a traffic-light in Kenmore Square, a busy crossroads in downtown Boston (MA, USA). A video camera co-located at the site recorded the vehicles passing the sensor, and this record was correlated with the PAS data. During a 5-day monitoring period (approximately 59 h) in the summer of 1998, over 34,000 motor vehicles were counted and classified and over 24,000 PAS readings were recorded (frequency = 1/8.6 s). The composition of the vehicle population was 94% passenger vehicles, 1.4% buses, 2.6% small trucks, 1.3% medium trucks, 0.35% large trucks, and 0.45% garbage and construction trucks. In analyzing the PAS data, it was assumed that the highest PAS measurements--those that exceeded the 95% critical level of the 5-min moving average of all the PAS measurements--were indicative of primary vehicular emissions. We found that approximately 46% of the mass of particle-bound PAH (i.e. approximately 46% of the integrated area under the PAS signal vs. time plots) was attributable to primary emissions from motor vehicles passing the sensor. Of this, 35-61% was attributable to passenger vehicles (cars, pickup trucks, and sports utility vehicles) and 39-65% was attributable to non-passenger vehicles [buses (14-23%), small trucks (12-20%), medium trucks (8.4-14%), large trucks (2.9-4.8%) and garbage and construction trucks (1.9-3.2%)]. Our results suggest that on a per vehicle basis, buses and trucks--the majority of which run on diesel fuel--emitted greater amounts of particle-bound PAH than passenger vehicles. Overall, we found that real-time photoelectric aerosol sensing (in combination with video photography) is useful for estimating the contributions of airborne PAB from different vehicle types. Due to the physical constraints of our monitoring site and the high volumes of traffic, however, it was not possible to uniquely attribute PAS signals to individual vehicles.

GnRH and gonadotropin release is decreased in chronic nitric oxide deficiency.

Nitric oxide synthetase (NOS), the conversion enzyme for nitric oxide (NO) is localized in the anterior pituitary of female rats, particularly in gonadotrophs and folliculo-stellate cells, suggesting that NO regulates the release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) from the anterior pituitary. The focus of this study was to determine the effect of chronic NO deficiency on the subsequent pituitary release of LH and FSH in vitro and the hypothalamic immunoexpression of GnRH in vivo. NO deficiency was induced by adding the NOS inhibitor, N-nitro-L-arginine (L-NNA, 0.6 g/L) to the drinking water of female Wistar rats. After 8 weeks, the animals were euthanized, the pituitaries were removed, and they were incubated in vitro. Pituitaries were perfused for 4 hr in the presence of pulsatile gonadotropin release hormone (GnRH, 500 ng/pulse) every 30 min. S-Nitroso-L-acetyl penicillamine (SNAP, an NO donor, 0.1 mM) or L-nitro-argine methyl ester (L-NAME, a NOS inhibitor, 0.1 mM) was added to the media and perfusate samples were collected at 10-min intervals. LH and FSH levels in the perfusate were measured by double antibody radioimmunoassays. Pituitaries from the NO-deficient rats had a significantly smaller GnRH-stimulated release of LH and FSH compared with proestrous control rats. The addition of S-NAP to the perfusate resulted in decreased LH and FSH secretion in the control group, but increased LH secretion in the NO-deficient group. The addition of L-NAME to the perfusate suppressed LH secretion from control pituitaries, but not in pituitaries from NO-deficient animals. Immunohistochemistry of brain slices demonstrated that NO-deficient rats had a large qualitative decrease of GnRH in the median eminence compared with their controls. This decrease was particularly evident in the external capillary plexus of the median eminence. We concluded that chronic NO deficiency is associated with a decreased GnRH in neurosecretory terminals in the external capillary layer of the median eminence, accompanied by a decrease in LH and FSH release from the pituitaries.

The effect of diabetes and sex on nitric oxide-mediated cardiovascular dynamics.

Diabetes is associated with impaired cardiovascular responses that are especially prominent in females. Since nitric oxide (NO)-mediated effects on cardiovascular dynamics are altered in diabetes, we evaluated the effect of L-NAME, a nitric oxide synthase (NOS) antagonist, on mean arterial pressure (MAP), heart rate (HR), and selective vascular flows in both male and female normal and diabetic rats as an index of NO activity. Rats were made diabetic using streptozotocin and maintained for 5-6 weeks. Following anesthesia with urethane/alpha-chloralose, the femoral artery and vein were cannulated for recording and sampling, and flow probes were placed on the iliac, renal, and superior mesenteric arteries. A bolus infusion of L-NAME (10mg/ kg) resulted in a rapid +52% and +68% increase in MAP in normal female and male rats, respectively. However, diabetic females' and males' responses were significantly lower (44% and 45%, respectively) when compared with their normal counterparts. The decreased HR in response to the peak pressor effect of L-NAME was more prominent in normal females compared with normal males (-14% vs 2%). The results in diabetic females and males were equivalent (-6% vs -9%, respectively). L-NAME decreased the conductance (flow/MAP) an average of 65% in all three vascular beds in normal female rats. In diabetic females, the iliac and superior mesenteric responses to L-NAME were less, and the renal conductance was contrastingly increased 23%. The response to L-NAME was comparable (-62%) in the renal and superior mesenteric and less (-40%) in the iliacs of normal versus diabetic males. We concluded that diabetes is associated with a decreased pressor response to NOS inhibition. And the impaired constriction response of the renal vessels noted in female diabetic rats may provide a basis for the increased renal pathology observed in diabetic humans.

Alteration of humoral and peripheral vascular responses during graded exercise in heart failure.

We hypothesized that performance of exercise during heart failure (HF) would lead to hypoperfusion of active skeletal muscles, causing sympathoactivation at lower workloads and alteration of the normal hemodynamic and hormonal responses. We measured cardiac output, mean aortic and right atrial pressures, hindlimb and renal blood flow (RBF), arterial plasma norepinephrine (NE), plasma renin activity (PRA), and plasma arginine vasopressin (AVP) in seven dogs during graded treadmill exercises and at rest. In control experiments, sympathetic activation at the higher workloads resulted in increased cardiac performance that matched the increased muscle vascular conductance. There were also increases in NE, PRA, and AVP. Renal vascular conductance decreased during exercise, such that RBF remained at resting levels. After control experiments, HF was induced by rapid ventricular pacing, and the exercise protocols were repeated. At rest in HF, cardiac performance was significantly depressed and caused lower mean arterial pressure, despite increased HR. Neurohumoral activation was evidenced by renal and hindlimb vasoconstriction and by elevated NE, PRA, and AVP levels, but it did not increase at the mildest workload. Beyond mild exercise, sympathoactivation increased, accompanied by progressive renal vasoconstriction, a fall in RBF, and very large increases of NE, PRA, and AVP. As exercise intensity increased, peripheral vasoconstriction increased, causing arterial pressure to rise to near normal levels, despite depressed cardiac output. However, combined with redirection of RBF, this did not correct the perfusion deficit to the hindlimbs. We conclude that, in dogs with HF, the elevated sympathetic activity observed at rest is not exacerbated by mild exercise. However, with heavier workloads, sympathoactivation begins at lower workloads and becomes progressively exaggerated at higher workloads, thus altering distribution of blood flow.

Leptin resistance in obesity is characterized by decreased sensitivity to proopiomelanocortin products.

Obesity in normal animals has been demonstrated to be associated with a decrease in sensitivity to leptin especially as it relates to leptin's capacity to increase sympathetic nerve activity and enhance cardiovascular dynamics. In normal animals leptin has been demonstrated to exert significant regulatory responses by its capacity to increase proopiomelanocortin (POMC) expression and especially the increase in alpha melanocyte stimulating hormone (alphaMSH). These responses to leptin are blocked by a melanocortin-4 (MC-4) receptor antagonist. In this study we investigated the responsiveness of the sympathetic nervous system and cardiovascular system of high fat fed obese animals to the intracerebroventricular (ICV) administration of the POMC products alphaMSH and beta-endorphin (beta-END). We further investigated these responses in obese animals following leptin administration in the presence of MC-4 receptor and opioid receptor blockade. The ICV administration of leptin resulted in an increase in lumbar sympathetic nerve activity (LSNA) and mean arterial pressure (MAP) in normals but decreased it in the obese. The ICV administration of alphaMSH increased the LSNA and MAP in normal animals but to a lesser degree in obese animals. On the other hand beta-endorphin decreased the LSNA and MAP in normal animals but increased it in obese animals. Additionally ICV leptin administration in obese animals in the presence of MC-4 or opioid receptor blockade resulted in an increase in sympathetic activity and a pressor response. From these studies we conclude that obesity in high fat fed animals is characterized by a decreased sensitivity to alphaMSH and a paradoxical response to beta-endorphin and this altered responsiveness may be a factor in the altered leptin resistance characteristic of obese animals.

Chronic intracerebroventricular insulin attenuates the leptin-mediated but not alpha melanocyte stimulating hormone increase in sympathetic and cardiovascular responses.

The co-existence of hyperinsulinemia and hyperleptinemia of obesity is well established. Additionally, both insulin resistance and leptin resistance are also characteristic of these states. Possible central nervous system (CNS) mechanisms could mediate these responses in that leptin receptors are located on hypothalamic neurons that coexpress neuropeptide-Y (NPY) or proopiomelanocortin (POMC) and both peptides that have been implicated as mediators of the CNS action of leptin. Leptin has been demonstrated to decrease or down regulate NPY expression and increase POMC expression. Insulin also has been demonstrated to decrease NPY and insulin insufficiency is associated with an increased POMC. Since both leptin and insulin share and modulate the same effector systems, we investigated the effect of CNS-induced hyperinsulinemia on the subsequent cardiovascular and sympathetic nervous response to leptin. Normal rats were implanted with intracerebroventricular (i.c.v.) cannula and allowed to recover. They were treated with insulin via i.c.v. cannula for 3 days. Following treatment, they were instrumented for the recording of cardiovascular and sympathetic nervous responses. Intracerebroventricular leptin administration in normal animals result in a progressive increase in both lumbar sympathetic nerve activity and mean arterial pressure. However, in animals pretreated with insulin for 3 days the leptin-induced response was completely attenuated. However, insulin treatment did not affect the POMC peptide product, alpha-melanocyte stimulating hormone (alphaMSH), mediated sympathetic nervous and cardiovascular responses. From these studies we conclude that CNS hyperinsulinemia can act to attenuate the leptin-induced increases in sympathetic nervous and cardiovascular system activity. The decreased responsiveness is not due to decreased sensitivity of the melanocortin, alphaMSH, mediated pathway.We suggest that the hyperinsulinemia of obesity may play a role in the obesity-induced leptin resistance.

Proopiomelanocortin (POMC) products in the central regulation of sympathetic and cardiovascular dynamics: studies on melanocortin and opioid interactions.

The proopiomelanocortin (POMC)-derived peptides are important regulators in a number of central nervous system pathways especially as they relate to food intake as well as metabolic and autonomic responses. In this study, we investigated the sympathetic nervous and cardiovascular responses to intracerebroventricular (i.c.v.) administration of alpha melanocyte stimulating hormone (alphaMSH), beta-endorphin (beta-END) and adrenal corticotrophic hormone (ACTH) alone or in the presence of a melanocortin antagonist, or an opioid antagonist, in normal animals. The i.c.v. administration of alphaMSH and ACTH resulted in a significant increase in the lumbar sympathetic nerve activity (LSNA) that was accompanied by an increase in mean arterial pressure (MAP). On the other hand i.c.v. administration of beta-END decreased the LSNA and MAP. The pretreatment of animals with the melanocortin-4 (MC-4) receptor antagonist, agouti protein, significantly antagonized the response to alphaMSH and also, paradoxically, not only antagonized the response to beta-END but converted its inhibitory responses on both the LSNA and MAP to a sympathetic activated and pressor response. Pretreatment with the opioid antagonist, naloxone, significantly antagonized the sympathetic nervous and cardiovascular response to beta-END. It partially but significantly antagonized the MAP response to alphaMSH, but the sympathetic response was unaffected. Neither agouti protein nor naloxone altered the sympathetic nervous and cardiovascular response to ACTH. From these studies, we conclude that i.c.v. administration of alphaMSH and ACTH increases the LSNA and cardiovascular dynamics, whereas beta-END decreases it. And, the MC-4 receptor antagonist reverses the endorphin response and the opioid antagonist attenuates the alphaMSH response suggesting possible receptor or central neural pathway interactions between MC-4 and the opioid receptor mediated effects.

Role and mechanism of action of C. PvuII, a regulatory protein conserved among restriction-modification systems.

The PvuII restriction-modification system is a type II system, which means that its restriction endonuclease and modification methyltransferase are independently active proteins. The PvuII system is carried on a plasmid, and its movement into a new host cell is expected to be followed initially by expression of the methyltransferase gene alone so that the new host's DNA is protected before endonuclease activity appears. Previous studies have identified a regulatory gene (pvuIIC) between the divergently oriented genes for the restriction endonuclease (pvuIIR) and modification methyltransferase (pvuIIM), with pvuIIC in the same orientation as and partially overlapping pvuIIR. The product of pvuIIC, C. PvuII, was found to act in trans and to be required for expression of pvuIIR. In this study we demonstrate that premature expression of pvuIIC prevents establishment of the PvuII genes, consistent with the model that requiring C. PvuII for pvuIIR expression provides a timing delay essential for protection of the new host's DNA. We find that the opposing pvuIIC and pvuIIM transcripts overlap by over 60 nucleotides at their 5' ends, raising the possibility that their hybridization might play a regulatory role. We furthermore characterize the action of C. PvuII, demonstrating that it is a sequence-specific DNA-binding protein that binds to the pvuIIC promoter and stimulates transcription of both pvuIIC and pvuIIR into a polycistronic mRNA. The apparent location of C. PvuII binding, overlapping the -10 promoter hexamer and the pvuIICR transcriptional starting points, is highly unusual for transcriptional activators.

The effect of the removal of the area postrema on insulin and IGF-1-induced cardiovascular and sympathetic nervous responses.

Previous studies have demonstrated that insulin and IGF-1 both increase lumbar sympathetic nerve activity (LSNA) and decrease mean arterial pressure (MAP). We hypothesized that the peripheral responses to insulin and IGF-1 are mediated, at least in part, via the central nervous system. In this study we determined the effects of the peripheral administration of both insulin and IGF-1 on cardiovascular dynamics and LSNA following removal of the area postrema (APX), a major site of blood-brain communication. Insulin infusion in normal rats decreased MAP but increased HR and LSNA. When insulin was infused in APX rats it also decreased the MAP but the MAP recovered rapidly and plateaued at a level equivalent to normals after 40 min. Insulin significantly increased the HR and LSNA in the APX rats compared to normals. However, when hypoglycemia was prevented by glucose infusion, the HR and LSNA responses to insulin in the APX rats were similar to normals. IGF-1 also decreased MAP and to a greater extent in the APX rats compared to normals but the increased LSNA in APX rats was equivalent to normals. The APX rats when compared to normals had a greater sensitivity to insulin-induced hypoglycemia while IGF-1 decreased the plasma glucose to a lesser degree in APX rats. We conclude that insulin and IGF-1 entry into the CNS at least via the area postrema does not contribute significantly to the hypotensive response and that the greater depressor response to IGF-1 is likely due to enhanced vascular sensitivity in APX rats. The increased HR and LSNA following insulin were likely mediated by an increased reflexive response to hypoglycemia.

Leptin-induced increase in sympathetic nervous and cardiovascular tone is mediated by proopiomelanocortin (POMC) products.

The mechanism underlying the leptin-induced increased sympathetic nerve activity and cardiovascular tone was investigated in normal rats. The melanocortin (MC) peptides and other fragments derived from proopiomelancortin (POMC) have a diverse array of biological activities and have been implicated in mediating the feeding behavioral responses to leptin. In this study we evaluated the possible involvement of two major products of POMC, alpha-melanocyte stimulating hormone (alpha-MSH) and beta-endorphin, in mediating the effects of leptin on sympathetic activity and mean arterial pressure (MAP) in normal rats. Intraventricular (i.c.v.) cannulas were implanted in normal rats and allowed to recover. On the day of the study the animals were anesthetized with urethane alpha-chloralose and instrumented for the recording of MAP, lumbar sympathetic nerve activity (LSNA), and heart rate (HR). To determine the correlation between the leptin response and the POMC products, alpha-MSH and beta-endorphins were also injected into the lateral ventricle. alpha-MSH acted to increase MAP and LSNA while beta-endorphin decreased these parameters. Leptin administration by i.c.v. cannula increased the MAP and LSNA in normal rats. The i.c.v. administration of agouti protein, an alpha-MSH receptor antagonist, prior to leptin infusion blocked this response. Likewise, pretreatment with naloxone a beta-endorphin receptor antagonist also blocked the response to leptin. From these studies we conclude that the acute increased LSNA and MAP in response to i.c.v. leptin may be mediated by increased POMC and its subsequent production of breakdown product alpha-MSH and/or beta-endorphin and it is the subsequent action of alpha-MSH that increases MAP and LSNA by activation of the MC4 receptor. The naloxone antagonism of the leptin response is likely due to the blockade of presynaptic opioid inhibition of the MC4 receptor-mediated pressor response.

Lipid peroxidation and antioxidant activities in rat testis after chronic cocaine administration.

OBJECTIVES: Our recent study has shown that cocaine has adverse action on spermatogenesis and fertility in male rats. Adverse effects of cocaine on the testes may be mediated by oxidative damage and subsequent lipid peroxidation. Glutathione is a cellular antioxidant and is found in high concentrations in the rat testes. In this study, the effects of chronic cocaine administration on the activities of glutathione peroxidase, the level of testicular reduced glutathione, and lipid peroxidation were investigated. METHODS: Thirty-day-old male Sprague-Dawley rats were given cocaine hydrochloride (15 mg/kg body weight) subcutaneously daily for 90 days. Control animals received equal volumes of normal saline daily for 90 days. Testes were removed at 15, 30, 60, and 90 days after cocaine injection. Tissues were washed and homogenized in ice-cold metaphosphoric acid solution or Tris-HCI buffer. Reduced glutathione, glutathione peroxidase, and malonaldehyde levels were determined by colorimetric assay. Statistical analysis was performed using analysis of variance. RESULTS: Testicular reduced glutathione and glutathione peroxidase were significantly decreased in the treatment testes 15, 30, 60, and 90 days after chronic cocaine injection compared with controls (P <0.05). The testicular malonaldehyde level was 20.8% (P <0.05), 22.1% (P <0.05), 31.2% (P <0.05), and 24.7% (P <0.05) above the control value on days 15, 30, 60, and 90, respectively. CONCLUSIONS: Our findings demonstrate that chronic administration of cocaine to male rats induces a depletion of reduced glutathione and glutathione peroxidase. Adverse effects of cocaine on the testes are at least in part due to impairment of the function of the antioxidant defense and further enhancement of lipid peroxidation.

Glycemic modulation of insulin/IGF-1 mediated skeletal muscle blood following sympathetic denervation in normal rats.

Both insulin and IGF-1 decrease vascular resistance and increase blood flow in skeletal muscle, and it has been suggested that the mechanistic action for insulin may be by increasing autonomic vasodilatory activity. In this study we evaluated the effects of insulin and IGF-1 on blood flow to denervated and non-denervated skeletal muscle as part of a continuing investigation into the mechanism of regulation of cardiovascular responses by these hormones. Normal rats were prepared for measurements of mean arterial pressure (MAP), heart rate (HR) and vascular flow in the left and right iliac artery. Resection of the left lumbar sympathetic chain increased flow (expressed as conductance, flow/MAP) in the denervated left iliac but not in the intact right artery. Subsequent insulin infusion where hypoglycemia was allowed to occur increased conductance in both arteries, but more so in the denervated artery. Similarly, IGF-1 infusion increased conductances in both intact and denervated iliac arteries, and the effect was slightly greater in the denervated artery. Insulin infusion when euglycemia was maintained increased conductance to a similar extent in denervated and intact iliac arteries. Contrastingly, IGF-1 infusion under euglycemic conditions resulted in a much greater increased conductance in the intact iliac. We conclude that both insulin and IGF-1 increase conductance directly and that glycemic status and sympathetic nerve activity modulate these responses. The insulin-induced increase in conductance in the denervated limb under hypoglycemic conditions suggest that hypoglycemic-stimulated epinephrine release may enhance the dilatory response. while the greater response to IGF-1 in the intact vessel under euglycemic conditions may be due to IGF-1 capacity to decrease sympathetic activity leading to an enhanced conductance.

Vascular dilatatory responses to sodium nitroprusside (SNP) and alpha-adrenergic antagonism in female and male normal and diabetic rats.

Diabetes is associated with impaired vascular dilatatory responses that appear to be influenced by sex as well as diabetic state. Therefore, we hypothesized that vascular and sympathetic control function exhibit a greater deterioration following the induction of diabetes in female than in male rats. We conducted a comparative determination of the effect of sodium nitroprusside (SNP, a nitrous oxide donor) and that of an alpha1-adrenergic antagonist, prazosin, on selective vascular flows, mean arterial pressure (MAP), and heart rate (HR), in female and male normal and diabetic rats. Rats were made diabetic using streptozotocin (50 mg/kg, iv) and maintained for 5-6 weeks. Following anesthesia with urethane/alpha-chloralose, the femoral artery and vein were cannulated for recording and sampling. Flow probes were placed on the iliac, renal, and superior mesenteric arteries. SNP (1, 5, 10, and 20 microg/kg) infusions resulted in a dose-dependent decrease in MAP in normal and diabetic rats. The decrease in MAP in normal males was 37% less at the 20 microg/kg concentration of SNP when compared to normal females. The HR was not significantly changed in response to the hypotensive effect of SNP; however, reflex tachycardia was more prominent in diabetic males. The vascular conductance (flow/MAP) was increased by SNP in normal and diabetic rats in a dose-dependent fashion; however, the responsiveness was decreased in the iliac and superior mesenteric and increased in the renal arteries in diabetics when compared to normals. Diabetic males were 42% and 28% less responsive to SNP in the iliac and superior mesenteric arteries, respectively. On the other hand, diabetic females were 1.5-fold more responsive in the renal artery when compared to normals. Prazosin (4 mg/kg) decreased the MAP in normal and diabetic rats to a comparable degree. Prazosin increased the vascular conductance in all three vascular beds in normal and diabetic rats with the greater increase occurring in the iliac (118%) and superior mesenteric (110%) arteries. We concluded that diabetes is associated with an increased response to NO in the renal vessels and a decreased response in the iliac and superior mesenteric vessels in both females and males. alpha-Adrenergic tone was greatest in diabetic female and male rats. This study suggests that decreased vascular flow in diabetes is a result of a combination of decreased sensitivity to NO and increased adrenergic tone.

Effects of general and selective beta-adrenergic antagonists on insulin-induced cardiac and selected vascular responses in rats.

Insulin administration results in vasodilation, decreased mean arterial blood pressure (MAP) and increased conductances (flow/MAP) in various vascular beds. beta-adrenergic blockers antagonize this response, but the mechanism of the interplay between insulin-induced vasodilation and beta-adrenergic antagonism is unknown. In this study, we evaluated the effects of beta-blockade using the selective beta(2) antagonist ICI 118551 or the general beta-antagonist propranolol on insulin-induced cardiac and regional flow responses in normal rats. Insulin-induced responses were also examined following adrenalectomy. Rats were anaesthetized and the femoral vein and artery were cannulated for infusions, sampling or monitoring of MAP and heart rate (HR). The iliac, renal, and superior mesentery arteries were equipped with pulsed-Doppler flow probes. Blood samples were collected at selected intervals. Insulin decreased blood glucose, MAP and increased conductances. Pretreatment with propranolol not only antagonized the insulin-induced decrease in MAP and increased conductance but insulin also then increased MAP and decreased conductances. ICI 11851, like propranolol, antagonized the insulin-induced decrease in MAP and increased iliac and renal artery conductances. Adrenalectomy did not alter the maximum insulin-induced effects on MAP and conductances but prevented the rebound recovery phase. beta-blockade following adrenalectomy had the same effects as beta-blockade alone on the insulin-induced responses. We conclude that the insulin-induced decrease in MAP and the increased flow in the selective vascular beds are modulated by a sympathetic beta(2)-receptor-mediated pathway and this response is not due primarily to the release of adrenal catecholamine.

Cocaine induced apoptosis in rat testes.

PURPOSE: Exposure of rats to chronic cocaine results in disruption of spermatogenesis including reduction of germ cells. However, the cellular mechanism responsible for the testicular damage in testes is still unknown. We have studied the role of apoptosis in cocaine induced testicular damage. MATERIALS AND METHODS: Thirty-day-old male Sprague-Dawley rats were given cocaine hydrochloride (15 mg./kg. body weight) subcutaneously daily for 90 days. Control animals received equal volumes of normal saline daily for 90 days. Testes were removed at 15, 30, 60, and 90 days of cocaine administration. In situ detection of germ cells with DNA strand breaks in paraffin-embedded testicular section (5 microm.) was achieved by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP in situ nick end-labeling (TUNEL) method. DNA fragmentation was also determined by gel electrophoresis. RESULTS: Apoptotic cells were found in the spermatocytes and spermatogonia of germinal epithelium. Less than 7% of seminiferous tubule cross sections showed a high level of apoptosis (> or =3 apoptotic cells per tubule) in control animals compared with experimental group where 25% of the tubules showed a high level of apoptosis (p<0.05). The number of apoptotic cells was significantly increased by 15 days, peaked at 30 days and persisted up to 90 days of cocaine exposure when compared with controls (p<0.05). DNA isolated from the cocaine treated testes displayed a clear ladder pattern whereas the DNA from controls did not. CONCLUSIONS: The experimental results presented here suggest that cocaine exposure leads to significant apoptosis in rat testes and the mechanism of cocaine induced testicular injury may be related to the induction of apoptosis.

Ovarian hormone secretory response to gonadotropins and nitric oxide following chronic nitric oxide deficiency in the rat.

Ovarian hormone secretion is regulated by gonadotropins, and it has been demonstrated that this response is modulated by nitric oxide (NO). The focus of this study was to determine the effect of chronic NO deficiency on the secretion of ovarian steroids. Female rats were given N-nitro-L-arginine (L-NNA; 0.6 g/L) in their drinking water, and vaginal smears were obtained daily. By 4 wk of treatment, all the rats were in constant estrus or proestrus. At 6-8 wk the animals were killed; the ovaries were removed and incubated in the presence of eCG (1 IU/ml) and hCG (1 IU/ml) and/or S-nitroso-L-acetyl penicillamine (an NO donor, S-NAP; 0.1 mM) for 4 h. Medium was collected at 30-min intervals, and estradiol, progesterone, and androstenedione were measured. Ovaries from proestrous rats served as controls. Ovaries from L-NNA-treated animals had a greater basal and gonadotropin-stimulated release of estradiol but not of androstenedione or progesterone in comparison to ovaries from untreated controls. S-NAP decreased the gonadotropin-stimulated estradiol, progesterone, and androstenedione in ovaries from NO-deficient rats. Steroid secretion in controls was not responsive to S-NAP. We conclude that chronic NO inhibition produces constant estrus due to increased estradiol production and that NO acts to inhibit estradiol and androstenedione production.

The co-existence of insulin-mediated decreased mean arterial pressure and increased sympathetic nerve activity is not mediated by the baroreceptor reflex and differentially by hypoglycemia.

In this study we measured simultaneously and sequentially the lumbar sympathetic nerve activity (LSNA) or renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP), and heart rate (HR) in response to insulin with co-existing hypoglycemia or with glucose replacement in normal rats. Sinoaortic denervation (SAD) was used to evaluate the influence of the baroreflex. LSNA, RSNA, MAP and HR were determined using an acquisition processor and computer software. Bolus insulin infusion where the blood glucose was allowed to decrease resulted in an immediate decrease in MAP. The HR decreased for approximately 15 min and subsequently increased. The LSNA increased immediately after insulin infusion peaking at 25 minutes and then recovered toward baseline. Insulin infusion with glucose replacement resulted in a decrease in MAP and HR. The LSNA progressively increased and was maintained throughout the experimental period. Insulin infusion with hypoglycemia increased RSNA and when hypoglycemia was prevented the RSNA decreased. SAD attenuated the decrease in MAP and LSNA response to insulin. Thus, insulin acts to decrease MAP while simultaneously increasing HR, LSNA and RSNA when hypoglycemia is allowed to occur. However, insulin acts to decrease HR and RSNA when euglycemia is maintained. The insulin-induced increase in LSNA is modulated by the baroreflex mechanism. We conclude that insulin has independent direct and indirect effects on LSNA, RSNA, MAP and HR that are modulated by glycemia and the baroreflex.

Regional blood flow dynamics in response to insulin and IGF-1 in diabetic animals.

Vascular changes in diabetes characterized by increased contractile or decreased dilator responses have been demonstrated in isolated blood vessels as well as in vivo systems. Previous studies in our laboratory have demonstrated that insulin and insulin like growth factor-1 (IGF-1) can decrease mean arterial pressure (MAP) and increase blood flow in vascular beds. In this study we evaluated the regional hemodynamic responses to insulin and IGF-1 in normal and diabetic rats. Normal male rats were made diabetic with streptozotocin (55 mg/kg) i.v. and maintained 60 to 70 days. On the day of the study the rats were anesthetized with urethane/chloralose, the femoral artery and vein cannulated for blood pressure monitoring and blood sampling or infusion, respectively. Pulsed-Doppler flow probes were placed around the iliac artery, renal artery and superior mesenteric artery to monitor blood flow. Insulin (16 nmol/kg) was infused as a bolus via the femoral vein and it decreased the MAP approximately 17% in both normal and diabetic rats. Insulin enhanced vascular flow (expressed as conductance) in the iliac and renal vascular bed but not the superior mesenteric vascular bed in normals. In diabetic rats the flow response to insulin compared to normals was attenuated in the iliac and renal vascular beds and increased in the superior mesenteric vascular bed. A bolus infusion of IGF-1 (16 nmol/kg) also decreased the MAP in normals and diabetics. IGF-1 increased vascular flow in all three vascular beds in normals but in the diabetics the response to IGF-1 was attenuated in the iliac, increased in the renal vascular bed and suppressed in the superior mesenteric vasculature. From these studies we conclude that diabetes is associated with an attenuated vascular response to insulin and IGF-1 in the iliac and renal vessels while insulin augments and IGF-1 decreases blood flow in the superior mesenteric vasculature.

The effect of glucose and calcium on Ca2+-adenosine triphosphatase in pancreatic islets isolated from a normal and a non-insulin-dependent diabetes mellitus rat model.

Regulation of calcium balance is important in the secretory function of pancreatic islets. Ca2+-adenosine triphosphatase (ATPase) is altered in tissues of non-insulin-dependent diabetes mellitus (NIDDM) rats, and they have an impaired response to glucose, "glucose blindness." We propose that the glucose blindness of the diabetic islet is the result of defective cellular calcium metabolism. Since Ca2+-ATPase activity is important in the regulation of calcium balance, we investigated the effect of glucose and/or calcium on Ca2+-ATPase activity in pancreatic islets in vitro and compared it with the effect in freshly isolated islets from controls and from rats with NIDDM induced by streptozotocin neonatally. Islets were isolated using collagenase and were stored fresh or cultured up to 2 days in RPMI 1640 in the presence of different concentrations of glucose and calcium. Membrane Ca2+-ATPase activity, insulin secretion, and insulin content were determined. Ca2+-ATPase activity was 1.30 +/- 0.20 micromol/L Pi/microg membrane protein in normal noncultured islets and 1.02 +/- 0.15 in islets cultured in 5.6 mmol/L glucose. Ca2+-ATPase activity progressively decreased to 0.56 +/- 0.10 and 0.34 +/- 0.14 micromol/L Pi/microg membrane protein when glucose was increased in the culture media to 16.6 and 27.7 mmol/L, respectively. Decreasing glucose to 2.8 mmol/L did not alter Ca2+-ATPase activity. Increasing or decreasing the Ca2+ content of the media did not significantly change Ca2+-ATPase activity. Islets isolated from NIDDM rats had lower basal Ca2+-ATPase activity and insulin content compared with normal controls. Incubation of islets from diabetic rats in high glucose further decreased the Ca2+-ATPase content, but incubation in low glucose did not reverse it. Insulin secretion was responsive to glucose and calcium in normal islets, but was suppressed in islets from diabetic animals. From these studies, we conclude that high glucose, but not calcium, decreases Ca2+-ATPase activity in islets from normal rats. Islets from NIDDM rats with glucose blindness have decreased Ca2+-ATPase activity, likely due to the glucose status. We suggest that this decreased Ca2+-ATPase activity may contribute to the pancreatic islets' glucose blindness.

Obesity due to high fat diet decreases the sympathetic nervous and cardiovascular responses to intracerebroventricular leptin in rats.

Obesity is associated with an increase in plasma leptin levels primarily derived from enhanced expression of the leptin gene in the adipose tissue. Leptin levels and expression are higher in females than males. The main functions of leptin are to decrease food intake and increase sympathetic nerve activity, especially in the brown adipose tissue. The high levels of leptin in obese, female rats suggest leptin resistance. In this article we describe experiments designed to investigate the effect of the intracerebroventricular (i.c.v.) administration of leptin on lumbar sympathetic nerve activity (LSNA) and cardiovascular parameters in female rats fed a low fat diet (control), a high fat diet (obese), or high fat diet followed by a period of food restrictions (reduced). The i.c.v. leptin administration increased LSNA in control rats, but decreased it in obese rats. In weight reduced animals the LSNA response to leptin returned to control levels. The i.c.v. leptin increased the mean arterial pressure in control and wt. reduced rats, but not in obese animals. The heart rate did not respond to leptin in any animal group. These results suggest that obesity decreases the central nervous system (CNS)-mediated lumbar sympathetic nervous and cardiovascular responses to leptin and that these responses recover following food restriction and wt. reduction. We conclude that obesity is associated with a decreased CNS response to leptin leading to a decrease in leptin effects to increase the activities of the autonomic nervous and cardiovascular systems.