Comparison of therapeutic regimens in the amelioration of alterations in rat gastrointestinal mucosal DNA, RNA and protein induced by streptozotocin diabetes mellitus.

Type 1 diabetes mellitus is characterized by hyperglycemia, insulinopenia, and secondary neural, renal and vascular complications. Clinical manifestations in the gastrointestinal tract range from initial mild complications to more severe complications as the disease progresses, but as of yet, are poorly understood. The current study has two main foci 1) to monitor the alterations in gastrointestinal DNA, RNA and protein content induced by streptozotocin diabetes and 2) to use these parameters to monitor the efficacy of intensive insulin treatment versus pancreatic islet transplantation in the amelioration of the diabetes induced alterations. Female Wistar Furth rats were rendered diabetic by streptozotocin injection and measured for alterations in gastrointestinal DNA, RNA and protein content. Similarly, animals which had streptozotocin-induced diabetes were also treated by intensive insulin therapy or pancreatic islet transplant and monitored for alterations in gastrointestinal DNA, RNA and protein content. In general, diabetes induced increases in stomach, duodenal, jejunal and colonic macromolecular content. With few exceptions, treatment with either intensive insulin or pancreatic islet transplantation returned each variable measured back to control levels. In every case, pancreatic islet transplantation was comparable to intensive insulin therapy. In the short term the treatments are comparable, but long term analyses are needed to determine if the treatments offer any difference in their ability to prevent the long term complications related to diabetes mellitus.

Pancreatic islet transplantation, but not intensive insulin therapy, corrects the pulmonary vascular complications of streptozotocin diabetes.

We examined the effects of long-term streptozotocin (STZ) diabetes and its treatment by intensive insulin therapy (IIT) or pancreatic islet transplantation on pulmonary pressor and depressor responses and segmental resistance profiles in female Wistar-Furth rats. Pulmonary vascular reactivity was examined using isolated, salt-perfused lungs at a constant flow rate of 30 mL.min-1.kg-1 body weight. Baseline perfusion pressure was significantly (p < 0.05) lower in lungs obtained from IIT animals compared with all other treatment groups. Following STZ administration, pressor responsiveness to 1.0 microgram of U-46619 (9,11-dideoxy-9 alpha, 11 alpha-methanoepoxy prostaglandin F2 alpha) was decreased in diabetic compared with IIT animals (9.33 +/- 0.54 vs. 11.94 +/- 0.29 mmHg (1 mmHg = 133.3 Pa)). Diabetes caused a similar decrease in the vasodilatory response to arginine vasopressin when compared with IIT animals (39.25 +/- 7.54 vs. 68.24 +/- 4.75%). Diabetes was also associated with a shift in the primary site of resistance from the pulmonary arterial to the pulmonary venous bed. This shift was restored to normal following pancreatic islet transplantation, but not IIT. IIT was also associated with significant alterations in the pattern of constrictor and dilator responses to U-46619 and arginine vasopressin. Pulmonary venous vasoconstrictor responses to U-46619 were augmented when compared with either control or diabetic animals, but not transplant. In addition, pulmonary venous vasoconstrictor responses in IIT animals were significantly greater than pulmonary arterial responses in the same group, a finding that was unique to IIT animals. Finally, IIT significantly augmented the pulmonary venous vasodilatory response to arginine vasopressin when compared with all other treatment groups. These data demonstrate significant alterations in pulmonary hemodynamic status of STZ diabetic female animals and suggest that pancreatic islet transplantation may be more beneficial than intensive insulin therapy in ameliorating these changes.

Acute nicotine pretreatment augments dopaminergic pulmonary vasodilation.

Nicotine use has been associated with augmented dopaminergic neurotransmission within the central nervous system by a number of researchers. We wished to determine if acute nicotine treatment would augment the pulmonary vasodilatory response to dopamine. Male Sprague-Dawley rats were pretreated with either subcutaneous nicotine or equivolume saline and a dose-response curve for dopaminergic pulmonary vasodilation was constructed ex vivo in isolated, salt-perfused rat lungs preconstricted with the synthetic thromboxane analogue U-46619. Nicotine pretreatment augmented the vasodilatory response to dopamine at doses falling within the mid range of the dopamine dose-response relationship, and this augmentation was blocked by the nicotinic ganglionic receptor antagonist mecamylamine. In contrast, nicotine did not augment the vasodilatory response produced by either the preferential DA1-dopaminergic receptor agonist SKF-38393 or the preferential DA2-dopaminergic receptor agonist quinpirole. Acute nicotine pretreatment did not affect the maximal pulmonary vasodilatory response produced by dopamine. Similarly, nicotine pretreatment failed to augment the vasodilatory response to the beta-adrenergic receptor agonists isoproterenol or terbutaline, but significantly diminished the response to dobutamine. Even though acute nicotine pretreatment did not augment beta-adrenergic receptor-mediated pulmonary vasodilation, the augmentation of dopaminergic responsiveness was inhibited by prior treatment with propranolol, suggesting beta-adrenergic receptor involvement. Finally, nicotine pretreatment did not alter the vasodilation produced by the nitric oxide dependent agents arginine vasopressin or sodium nitroprusside. These data demonstrate that nicotine alters dopaminergic vasodilation in the pulmonary vascular bed.

Differential pulmonary vascular effects of streptozotocin diabetes in male and female rats.

We examined the effect of streptozotocin (STZ) diabetes on pulmonary pressor responses and segmental pulmonary vascular resistance in male and female Wistar-Furth rats. Pulmonary vascular reactivity was studied using isolated, salt-perfused lungs at a constant flow rate of 30 ml/min/kg body weight. Following STZ administration, pressor responsiveness to 1.0 microg of U-46619 (9,11 dideoxy-9alpha, 11alpha-methanoepoxy Prostaglandin F2alpha) was diminished (p < 0.05) in lungs obtained from male diabetic rats when compared to sham treated controls (7.87 +/- 1.67 vs. 13.59 +/- 1.67 mmHg). In contrast, diabetes failed to affect pressor responsiveness in lungs from female animals. In another set of animals, segmental pulmonary vascular resistance was determined in lungs isolated from male and female diabetic or sham-treated (STZ carrier vehicle) animals. Total pulmonary vascular resistance was significantly elevated in male diabetic animals as compared to controls. This elevation was attributable to significant increases in resistance at the level of small pulmonary veins. In addition, diabetes was associated with a shift in the primary site of resistance from small arteries to small veins in male animals. We were unable to detect any effect of short-term diabetes on the segmental resistance profile in lungs obtained from female animals. These data indicate that both the pulmonary segmental resistance profile and pulmonary vascular reactivity are altered by short-term diabetes in male rats. Additionally, these studies demonstrate gender-related effects of short-term diabetes, which may suggest a more favorable pulmonary response to diabetes in female animals.

Pulmonary circulation demonstration using an isolated rat lung model.

We have developed a pulmonary circulation laboratory exercise that effectively illustrates basic concepts typically taught in a graduate physiology curriculum. The demonstration uses an isolated, perfused rat lung model to delineate the mechanisms by which pulmonary vascular resistance can be altered either passively or in an active manner by contraction or relaxation of vascular smooth muscle. The exercise further offers an opportunity to closely observe an experimental preparation commonly used to study the pulmonary circulation and allows students the opportunity to interpret the resulting physiological data. Student evaluations indicate that the demonstration was received with enthusiasm and provides an effective teaching tool for reinforcing concepts in pulmonary vascular physiology.

Alteration of segmental vascular resistance in the pulmonary circulation of diabetic rats.

Numerous studies have shown that altered plasma glucose and (or) insulin have profound effects on the pulmonary circulation. Recently, we documented diminished pressor responses to the synthetic thromboxane analogue U-46619 (9,11-dideoxy-9 alpha, 11 alpha-methanoepoxy prostaglandin F2 alpha) following short-term streptozotocin-induced diabetes in rats. However, these earlier studies examined the effects of diabetes on resistance changes across the entire pulmonary vasculature and made no effort to localize the site(s) of any abnormalities. Thus, in the present study we examined segmental pulmonary vascular resistances using the double-occlusion method in isolated, perfused rat lungs 2 weeks after streptozotocin-induced diabetes. Under baseline conditions, total pulmonary vascular resistance (TPVR) did not differ in lungs isolated from control and diabetic animals (0.66 +/- 0.03 vs. 0.85 +/- 0.05 mmHg.mL-1.min-1, respectively (1 mmHg = 133.3 Pa)). However, control animals demonstrated greater arterial (Ra) than venous (Rv) contribution to TPVR (0.43 +/- 0.02 vs. 0.23 +/- 0.02 mmHg.mL-1.min-1, respectively). This relationship was reversed in diabetic animals (Ra = 0.30 +/- 0.02 mmHg.mL-1.min-1; Rv = 0.54 +/- 0.04 mmHg.mL-1.min-1). Following constriction with U-46619 this pattern persisted, although the absolute vasoconstrictor response to the agent was similar in each segment. Likewise, this pattern of resistance was unaffected following dilation of the pulmonary vascular bed with arginine vasopressin. These findings illustrate that pulmonary segmental vascular resistances are altered and, more specifically, that pulmonary venous resistance is selectively increased, 2 weeks following the induction of diabetes.

Cardiovascular responses to hemorrhage during acute and chronic hypoxia.

Previous work from our laboratory had demonstrated attenuation of systemic vasoreactivity to pressor agents in rats after acute or chronic exposure to hypoxia. Therefore we hypothesized that hemorrhage of acutely hypoxic (12% O2) or chronically hypoxic (barometric pressure 380 mmHg for 3 wk) rats would deter the normal increase in total peripheral resistance (TPR) and thus decrease the ability to maintain blood pressure. Progressive hemorrhage (2% of blood volume per min) was performed under conditions of either normoxia or acute hypoxia in conscious rats. In control animals, the increase in TPR observed during normoxic hemorrhage was absent when hemorrhage was performed in acute hypoxia. Furthermore, the amount of blood removal required to achieve hypotension was reduced under conditions of acute hypoxia. In contrast, chronically hypoxic rats exhibited no difference in the threshold for hypotension between conditions of acute normoxia and hypoxia and demonstrated an increased hypotensive threshold under both normoxic and hypoxic conditions compared with control animals. We next hypothesized that the prolonged threshold for hypotension observed in chronically hypoxic rats might be due to hypoxia-induced right ventricular hypertrophy. Such ventricular hypertrophy may minimize stimulation of ventricular volume receptors thought to elicit the reflex fall in heart rate and TPR occurring in extreme underfill conditions. Therefore we compared the cardiovascular responses to hemorrhage in rats with right ventricular hypertrophy resulting from administration of monocrotaline with those from rats treated with vehicle.(ABSTRACT TRUNCATED AT 250 WORDS)

Potassium channels are not involved in vasopressin-induced vasodilation in the rat lung.

We have previously observed that arginine vasopressin (AVP)-induced pulmonary vasodilation is attenuated by nitric oxide (NO) synthesis inhibition; however, blockade of the response is incomplete even at very high doses of the inhibitor. Thus it was hypothesized that the remaining vasodilation might be due to release of an endothelium-derived hyperpolarizing factor acting to open vascular smooth muscle K+ channels. Lungs were isolated from male Sprague-Dawley rats and perfused at constant flow with physiological saline solution containing 4% albumin. After equilibration, lungs were treated with either glibenclamide (50 microM), Ba2+ (100 microM), tetraethylammonium (10 mM), or the respective vehicle and were then constricted with the thromboxane mimetic U-46619. Upon development of a stable degree of vasoconstriction, AVP (2.5 x 10(-9) M) was administered and its vasodilator action noted. AVP caused an approximately 60% reversal of U-46619 vasoconstriction in control lungs, and this response was not affected by any of the K+ channel blockers. In contrast, administration of the NO synthesis inhibitor N omega-nitro-L-arginine (L-NNA; 300 microM) significantly attenuated AVP-induced dilation to approximately 25%. The addition of K+ channel blockers did not further diminish the vasodilatory response in L-NNA-treated lungs. In conclusion, these results suggest that ATP- and Ca(2+)-sensitive K+ channels are not involved in the pulmonary vasodilatory response to AVP.

Oxytocin augments baroreflex bradycardia in conscious rats.

Previous studies have demonstrated augmentation of baroreflex-mediated bradycardia by arginine vasopressin (AVP). However, the specific receptor subtype responsible for mediating this augmentation has not been determined. In the present study, experiments were performed in conscious rats to determine the possible involvement of oxytocin receptors in this response. Infusion of oxytocin at a dose that had no effect on baseline hemodynamic values significantly augmented the bradycardic response to IV bolus doses of methoxamine. Prior treatment with selective antagonists to either oxytocin, V1 vasopressinergic or V2 vasopressinergic receptors reversed this enhancement. In a separate set of experiments, baroreflex-mediated bradycardic responses to IV bolus doses of AVP were assessed. Pretreatment with the selective oxytocin receptor antagonist reversed vasopressinergic augmentation of baroreflex sensitivity. Finally, combined vasopressinergic and oxytocinergic stimulation of the baroreflex was assessed. Treatment with both AVP and oxytocin did not augment baroreflex-mediated bradycardia greater than AVP alone. We conclude from these experiments that AVP and oxytocin both augment baroreflex sensitivity, although the receptor type(s) responsible are not clear.

Maintained endothelium-dependent pulmonary vasodilation following chronic hypoxia in the rat.

We have previously demonstrated that arginine vasopressin (AVP) dilates the preconstricted pulmonary vasculature via the release of nitric oxide (NO). However, recent evidence suggests that NO release in response to other agents may be suppressed in lungs from animals that have been chronically exposed to hypoxia. The purpose of the present experiment was to determine whether vasopressinergic pulmonary vasodilation is similarly affected by chronic exposure to hypoxia (barometric pressure = 380 Torr for 4 wk). Inhibition of NO synthesis with N omega-nitro-L-arginine (L-NNA) had no effect on baseline perfusion pressure in isolated salt-perfused lungs from either control or chronically hypoxic rats. Similarly, pulmonary vasodilatory responses to AVP and the calcium ionophore A23187 were unaffected by chronic hypoxic exposure. Pretreatment with the cyclooxygenase inhibitor meclofenamate did not alter vasopressinergic pulmonary vasodilation in lungs from either control or chronically hypoxic animals, ruling out involvement of vasodilator prostaglandins in the response to AVP. In contrast, vasodilatory responses to both AVP and A23187 were inhibited by L-NNA pretreatment not only in lungs from control animals but also in lungs from chronically hypoxic rats, suggesting the involvement of NO in the vasodilatory response. The inhibition by L-NNA was reversible by prior addition of excess L-arginine but not by D-arginine. In addition, vasodilatory responses to the endothelium-independent vasodilators sodium nitroprusside and isoproterenol were unaffected by chronic hypoxic exposure. We conclude that endothelium-dependent vasodilation remains intact in male Sprague-Dawley rats after chronic hypoxic exposure.

Pulmonary vasodilatory response to neurohypophyseal peptides in the rat.

Experiments were performed on isolated salt-perfused rat lungs to determine the receptor type(s) responsible for the pulmonary vascular effects of the neurohypophyseal peptides arginine vasopressin (AVP) and oxytocin. Bolus administration of AVP to lungs preconstricted with the thromboxane mimetic U-46619 resulted in a dose-dependent vasodilatory response (approximately 65% reversal of U-46619-induced vasoconstriction at the highest dose tested) that was blocked by pretreatment with a selective V1- but not by a selective V2-vasopressinergic receptor antagonist. Administration of a selective V1-agonist to the preconstricted pulmonary vasculature resulted in a vasodilatory response similar to that observed with AVP (approximately 55% reversal of U-46619 vasoconstriction), which was blocked by prior administration of the selective V1-receptor antagonist. Administration of the selective V2-receptor agonist desmopressin to the preconstricted pulmonary vasculature resulted in a small (approximately 8% reversal of U-46619 vasoconstriction) vasodilatory response that was, nevertheless, greater than that produced by addition of vehicle alone and was attenuated by pretreatment with a selective V2-receptor antagonist. Finally, oxytocin also caused vasodilation in the preconstricted pulmonary vasculature; however, the potency of oxytocin was approximately 1% of AVP, and the vasodilation produced by oxytocin was blocked by prior administration of a selective V1-receptor antagonist, suggesting that oxytocin acts via V1-vasopressinergic receptor stimulation. We conclude from these experiments that AVP and oxytocin dilate the preconstricted pulmonary vasculature primarily via stimulation of V1-vasopressinergic receptors. V2-receptor stimulation results in a minor vasodilatory response, although its physiological significance is unclear.

Augmentation of baroreflex-mediated bradycardia in conscious pregnant rats.

The goal of the present study was to examine baroreflex control of heart rate during pregnancy in chronically instrumented unrestrained rats. The same rats (n = 6) were studied before conception, again on gestational days 5, 12, and 19, and last on postpartum day 6; thus each rat served as its own control. Time control experiments were also conducted in a separate group of virgin rats (n = 7). Resting mean arterial pressure decreased by 10 mmHg on gestational day 19 (P less than 0.01 vs. prepregnant), and heart rate significantly increased by approximately 10% relative to time control rats. Dose-response curves were constructed for methoxamine and sodium nitroprusside comparing the various dosages with systemic pressor and depressor responses, respectively. The dose-response relationship for methoxamine was shifted to the right in gravid rats of 19 gestational days (P less than 0.03 vs. prepregnant), indicating an attenuation of alpha-adrenergic receptor-mediated pressor responsiveness. In contrast, depressor responses to sodium nitroprusside were not significantly altered in pregnancy. Baroreflex-mediated bradycardia was unchanged until gestational day 19, when enhanced bradycardia responses to methoxamine were observed. Baroreflex-mediated tachycardic responses elicited by sodium nitroprusside were not affected at any stage of pregnancy. Baroreflex control of heart rate did not change significantly with either increases or decreases of blood pressure in time control experiments. We conclude that during late pregnancy in conscious rats 1) resting blood pressure decreases and heart rate increases, 2) systemic pressor responses to methoxamine are diminished, and 3) baroreflex-mediated bradycardia is enhanced.

Role of nitric oxide in vasopressinergic pulmonary vasodilatation.

Experiments were performed to determine the mechanism of vasopressinergic pulmonary vasodilation in isolated, salt-perfused rat lungs. Administration of a 50-ng bolus of arginine vasopressin (AVP) to lungs preconstricted with the synthetic thromboxane analogue U-46619 resulted in a 66% reversal of pulmonary vasoconstriction. Administration of the known endothelium-dependent vasodilator ATP resulted in a parallel decrease in pressure. The vasodilatory responses to both agents were significantly attenuated by pretreatment with the nitric oxide synthesis inhibitor N omega-nitro-L-arginine (L-NNA). In addition to attenuating the vasodilatory response to these agents, L-NNA pretreatment caused a significant augmentation of the pressor response to U-46619 without affecting baseline pulmonary arterial pressure. The attenuation of vasopressinergic pulmonary vasodilation by L-NNA was completely reversed by addition of excess substrate for NO production (50 mM L-arginine) but was unaffected by addition of equimolar amounts of D-arginine. Finally, L-NNA pretreatment failed to attenuate the vasodilatory actions of sodium nitroprusside and isoproterenol. We conclude that AVP dilates the preconstricted pulmonary vasculature via the release of nitric oxide.

Role of vasopressin in cardiovascular responses to acute and chronic hyperosmolality.

Experiments were performed in conscious chronically instrumented rats to determine the role of arginine vasopressin (AVP) in the cardiovascular adjustments to acute and chronic increases in plasma osmolality. Animals were implanted with pulsed Doppler flow probes and arterial and venous catheters for the determination of cardiac output, mean arterial blood pressure (MABP), and heart rate and for the calculation of total peripheral resistance and baroreflex sensitivity (BRS). Before and after raising plasma osmolality by either 48-h water deprivation or acute hypertonic saline infusion, specific V1- or V2-vasopressinergic receptor antagonists or vehicle were administered to the animals, and the cardiovascular responses were noted. MABP was significantly elevated in water-deprived animals. These animals also exhibited significantly increased BRS, which was further increased by administration of the V1-receptor antagonist. Animals subjected to acute hypertonic saline infusion also demonstrated increased MABP, although the infusion, unlike water deprivation, did not affect BRS. We observed no significant effects on any other variable measured. We conclude that AVP plays a relatively minor role in the cardiovascular adjustments to acute and chronic hyperosmolality.

Role of vasopressin in acutely altered baroreflex sensitivity during hemorrhage in rats.

Experiments were performed to examine the potential role of circulating arginine vasopressin (AVP) on baroreflex sensitivity during hypotensive and nonhypotensive hemorrhage in the conscious rat. Animals were chronically instrumented for measurement of cardiac output, blood pressure, and heart rate (HR). Three potential stimuli for release of AVP were utilized: 1) rapid 20% arterial hemorrhage that resulted in hypotension, 2) nonhypovolemic hypotension induced by intravenous infusion of nitroprusside, and 3) nonhypotensive hemorrhage (rapid 10% arterial blood withdrawal). Hypotensive hemorrhage was associated with significant reductions in blood pressure, cardiac output, HR, and calculated total peripheral resistance, an increase in baroreflex (BRR) bradycardia in response to pressor infusions of phenylephrine, and a moderate elevation in circulating AVP. Prior intravenous administration of a specific V1-vasopressinergic antagonist augmented the hypotensive response to hemorrhage; however, neither V1- nor V2-blockade affected hemorrhage-induced augmentation of the BRR. Inducement of hypotension by infusion of nitroprusside did not alter subsequent BRR sensitivity. Finally, nonhypotensive hemorrhage was associated with an increase in resting HR and augmented BRR sensitivity. However, in contrast to hypotensive hemorrhage, either V1- or V2-antagonism attenuated the increase in BRR sensitivity seen with 10% hemorrhage. These data suggest that, although AVP may play a role in blood pressure maintenance via its direct vasoconstrictor actions during hypotensive hemorrhage, the observed augmentation of BRR sensitivity associated with severe blood loss is not attributable to a vasopressinergic mechanism activated by circulating AVP. However, blood-borne AVP may contribute to BRR sensitivity alterations in response to mild blood loss.

Ethanol exhibits alpha receptor blocking-like properties in anesthetized rats.

The ability of ethanol to reduce alpha-adrenergic receptor-mediated pressor responsiveness in vivo was investigated in chloralose-anesthetized male Sprague-Dawley rats. Catheters were inserted in the jugular vein and the femoral artery of rats for the injection of drugs and the measurement of blood pressure, respectively. Dose-response curves for phenylephrine and norepinephrine were constructed by plotting the change in mean arterial pressure following a bolus dose of the agent against the dose of the pressor agent used. Following construction of an initial dose-response curve, animals were challenged with either a 1 g/kg dose of ethanol or an equivalent volume of saline (iv) and the dose-response curves were repeated. Using a similar protocol, pressor responsiveness was evaluated in animals pretreated with either yohimbine (1 mg/kg) or prazosin (3.9 micrograms/kg), a dose sufficient to produce partial blockade of alpha receptor-mediated pressor responsiveness, and then treated with ethanol. Ethanol produced a partial blockade of alpha receptors when the animals were challenged with either phenylephrine or norepinephrine. This blockade produced by ethanol was shown to be similar to that produced by the receptor blocking agents used in this study. To rule out any nonspecific effects of ethanol in reducing vascular reactivity, some animals were challenged with angiotensin II both before and after treatment with ethanol, yohimbine, or prazosin and after both drugs were administered together. Ethanol, as well as the alpha 1- and alpha 2-adrenergic blocking agents tested failed to have any significant effect on angiotensin II-pressor responsiveness, ruling out any nonspecific effect of ethanol on the vasculature. It is concluded, therefore, that ethanol has alpha receptor blocking-like activity in vivo.

Prenatal ethanol exposure in rats does not alter maze exploration or impair visual discrimination with or without distracting stimuli.

The effects of in utero exposure to ethanol on maze exploration and visual discrimination and reversal with and without distracting tactile stimuli present were examined in rats. Three groups were compared on all measures. An ethanol treated group was the offspring of dams receiving a liquid diet throughout pregnancy with 35% of the calories supplied as ethanol. Subjects in a pair-fed group were the offspring of dams fed the same diet except that the caloric equivalent of maltose-dextrin was substituted for the ethanol. Subjects in a third group were the offspring of dams fed ad lib lab chow and water throughout pregnancy. All pups were nursed by foster mothers who received ad lib lab chow and water throughout pregnancy and nursing. Relative to the control groups, the ethanol treated group was not found to be significantly more active in the maze at 41-45 days of age or deficient in visual discrimination or reversal with or without the distracting stimuli present at 46-77 days of age. These results are consistent with many studies suggesting that behavioral deficits resulting from in utero ethanol exposure become less evident with age.