Development of the Physiology Professional Skills Curriculum Mapping Tool (PS-MAP).

During the course of undergraduate studies, physiology (and related STEM) majors should acquire a both broad and in-depth foundation in physiological knowledge along with a distinct range of transferable (professional) skills (e.g., critical thinking, communication skills, data analysis). Previously, through a consultative and iterative process with physiology educators, the Professional Skills Committee of the Physiology Majors Interest Group (PMIG) defined and refined a consensus list of professional skills that physiology majors should acquire during their program of study. Here we describe the development and beta testing of a convenient tool to enable physiology and physiology-related program educators to map these professional skills across their curricula. The tool, referred to as PS-MAP, uses the Qualtrics platform and allows programs to collect and organize data about whether students are provided the opportunity to learn and develop the defined professional skills during their undergraduate experience. The authors have made the PS-MAP tool freely available to educators and provide practical tips for its implementation. Use of the PS-MAP tool and the data collected can help programs identify curricular strengths and gaps as well as facilitate curricular discussions among educators within the program.NEW & NOTEWORTHY In addition to foundational physiology knowledge, undergraduate physiology and related STEM majors should develop a range of transferable professional skills. However, evidence of this curricular goal has been lacking. Therefore, the Professional Skills Committee of the Physiology Majors Interest Group (PMIG) developed the freely available and convenient Physiology Professional Skills Curriculum Mapping Tool (PS-MAP) to assist educators in mapping these professional skills throughout their programs.

Effects of active and passive land use management after cropland abandonment on water and vegetation dynamics in the Central Spanish Pyrenees.

The Mediterranean mountains have been subject to significant land abandonment process during the second half of the 20th century. The subsequent natural revegetation following abandonment in rural areas has been widely documented to have substantial implications on the hydrological cycle and the vegetation. The Spanish Pyrenees are one of the most affected areas by these land transformations which could threaten their importance for water supply and agricultural activities in the downstream lowland areas. Land managers as well as scientists around the world have taken different positions on how to deal with these land use changes. Some are in favor of active management (AM) (i.e. density reduction) while others are supporting passive management (PM) (letting the process of revegetation continue). This study aims to investigate the implication of AM and PM on hydrological and vegetation dynamics under different climate trajectories in a representative abandoned cropland catchment in the Central Spanish Pyrenees. A coupled ecohydrologic model is used to estimate the post management response of streamflow (STR), evapotranspiration (ET), soil saturation deficit (SD) and plant carbon (PC) following shrub clearing. Clearing increased annual STR by 16%, while ET and SD decreased by around -9% and -6% respectively during the first year after management with changes to monthly flows. These changes to water regimes may be even higher in wetter years. Over a 10-years period of vegetation recovery annual STR increased between 7.1% and 24.2%, while annual ET and SD decreased between -2.6% to -8.7% and -2.7% to -6% respectively due to shrub clearing, with the highest changes occurring in the first three years of AM. On the effect of climate change, our results show that a 2 °C increase in temperature could reduce AM effects on water regimes and accelerate the recovery of PC given averaged rainfall conditions.

Sympathetic control of heart rate in nNOS knockout mice.

Inhibition of neuronal nitric oxide synthase (nNOS) in cardiac postganglionic sympathetic neurons leads to enhanced cardiac sympathetic responsiveness in normal animals, as well as in animal models of cardiovascular diseases. We used isolated atria from mice with selective genetic disruption of nNOS (nNOS(-/-)) and their wild-type littermates (WT) to investigate whether sympathetic heart rate (HR) responses were dependent on nNOS. Immunohistochemistry was initially used to determine the presence of nNOS in sympathetic [tyrosine hydroxylase (TH) immunoreactive] nerve terminals in the mouse sinoatrial node (SAN). After this, the effects of postganglionic sympathetic nerve stimulation (1-10 Hz) and bath-applied norepinephrine (NE; 10(-8)-10(-4) mol/l) on HR were examined in atria from nNOS(-/-) and WT mice. In the SAN region of WT mice, TH and nNOS immunoreactivity was virtually never colocalized in nerve fibers. nNOS(-/-) atria showed significantly reduced HR responses to sympathetic nerve activation and NE (P < 0.05). Similarly, the positive chronotropic response to the adenylate cyclase activator forskolin (10(-7)-10(-5) mol/l) was attenuated in nNOS(-/-) atria (P < 0.05). Constitutive NOS inhibition with L-nitroarginine (0.1 mmol/l) did not affect the sympathetic HR responses in nNOS(-/-) and WT atria. The paucity of nNOS in the sympathetic innervation of the mouse SAN, in addition to the attenuated HR responses to neuronal and applied NE, indicates that presynaptic sympathetic neuronal NO does not modulate neuronal NE release and SAN pacemaking in this species. It appears that genetic deletion of nNOS results in the inhibition of adrenergic-adenylate cyclase signaling within SAN myocytes.

NOS isoform-specific regulation of basal but not exercise-induced mitochondrial biogenesis in mouse skeletal muscle.

Nitric oxide is a potential regulator of mitochondrial biogenesis. Therefore, we investigated if mice deficient in endothelial nitric oxide synthase (eNOS-/-) or neuronal NOS (nNOS-/-) have attenuated activation of skeletal muscle mitochondrial biogenesis in response to exercise. eNOS-/-, nNOS-/- and C57Bl/6 (CON) mice (16.3 +/- 0.2 weeks old) either remained in their cages (basal) or ran on a treadmill (16 m min(-1), 5% grade) for 60 min (n = 8 per group) and were killed 6 h after exercise. Other eNOS-/-, nNOS-/- and CON mice exercise trained for 9 days (60 min per day) and were killed 24 h after the last bout of exercise training. eNOS-/- mice had significantly higher nNOS protein and nNOS-/- mice had significantly higher eNOS protein in the EDL, but not the soleus. The basal mitochondrial biogenesis markers NRF1, NRF2alpha and mtTFA mRNA were significantly (P< 0.05) higher in the soleus and EDL of nNOS-/- mice whilst basal citrate synthase activity was higher in the soleus and basal PGC-1alpha mRNA higher in the EDL. Also, eNOS-/- mice had significantly higher basal citrate synthase activity in the soleus but not the EDL. Acute exercise increased (P< 0.05) PGC-1alpha mRNA in soleus and EDL and NRF2alpha mRNA in the EDL to a similar extent in all genotypes. In addition, short-term exercise training significantly increased cytochrome c protein in all genotypes (P< 0.05) in the EDL. In conclusion, eNOS and nNOS are differentially involved in the basal regulation of mitochondrial biogenesis in skeletal muscle but are not critical for exercise-induced increases in mitochondrial biogenesis in skeletal muscle.

Changes in acid precipitation-related water chemistry of lakes from southwestern New Brunswick, Canada, 1986-2001.

Between 1986 and 2001, thirty-nine lakes in southwestern New Brunswick in Atlantic Canada were surveyed for acid precipitation-related water quality changes. Most of the study lakes are located on granite bedrock and represent the most acid sensitive lakes in the province. Between 1987 and 1992, hydrogen ion deposition to the lake study area averaged 452 eq ha(-1) yr(-1), compared to 338 eq ha(-1) yr(-1) between 1993 and 2000, a 25% reduction. The lake chemistry data were evaluated by dividing the lakes into four clusters for each survey year based on their acid neutralizing capacity. Twenty percent of the lakes (cluster IV) had an average ANC of 40 microeq L(-1) or greater and maintained an average pH of greater than 6 over the duration of the study period. A pH of 6 or greater is considered a healthy benchmark for maintaining biodiversity. The remaining 31 lakes (clusters I to III) had an average ANC of less than 40 microeq L(-1) and maintained an average pH of less than 6. Other lake chemistry changes included a general decline in lake sulphate and colour over the duration of the survey period, followed by more recent improvements in calcium ion, pH and ANC, and notably higher but declining aluminum levels in lower ANC and pH lakes. Nitrate accounted for 37% of the acid deposition to the study area, however it was not detectable in the lakes. Although acid deposition has declined and these lakes are beginning to show signs of acid recovery, 80% of the study lakes remain acid sensitive having little buffering capacity with low calcium, pH and ANC.

Neuronal control of heart rate in isolated mouse atria.

A novel mouse isolated atrial preparation with intact postganglionic autonomic innervation was used to investigate the neuronal control of heart rate. To establish whether autonomic activation was likely to alter heart rate by modulating the hyperpolarization-activated current (If), the L-type Ca2+ current (ICa,L), or the ACh-activated K+ current (IK,ACh), the effects of nerve stimulation (right stellate ganglion or right vagus, 1-30 Hz) and autonomic agonists (0.1 microM norepinephrine or 0.3 microM carbachol) on heart rate were investigated in the presence of inhibitors of these currents, cesium chloride (Cs+, 1 mM), nifedipine (200 nM), and barium chloride (Ba2+, 0.1 mM), respectively. The positive chronotropic response to stellate ganglion stimulation was reduced by approximately 20% with Cs+ and nifedipine (P < 0.05), whereas the heart rate response to norepinephrine was only reduced with Cs+ (P < 0.05). Ba2+ attenuated the decrease in heart rate with vagal stimulation and carbachol by approximately 60% (P < 0.05). These results are consistent with the idea that sympathetic nerve stimulation modulates If to increase heart rate in the mouse. Activation of ICa,L also appears to contribute to the sympathetic heart rate response. However, the decrease in heart rate with vagal stimulation or carbachol is likely to result primarily from the activation of IK,ACh.

Peripheral vagal control of heart rate is impaired in neuronal NOS knockout mice.

The role of nitric oxide (NO) in the vagal control of heart rate (HR) is controversial. We investigated the cholinergic regulation of HR in isolated atrial preparations with an intact right vagus nerve from wild-type (nNOS+/+, n = 81) and neuronal NO synthase (nNOS) knockout (nNOS-/-, n = 43) mice. nNOS was immunofluorescently colocalized within choline-acetyltransferase-positive neurons in nNOS+/+ atria. The rate of decline in HR during vagal nerve stimulation (VNS, 3 and 5 Hz) was slower in nNOS-/- compared with nNOS+/+ atria in vitro (P < 0.01). There was no difference between the HR responses to carbamylcholine in nNOS+/+ and nNOS-/- atria. Selective nNOS inhibitors, vinyl-L-niohydrochloride or 1-2-trifluoromethylphenyl imidazole, or the guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one significantly (P < 0.05) attenuated the decrease in HR with VNS at 3 Hz in nNOS+/+ atria. NOS inhibition had no effect in nNOS-/- atria during VNS. In all atria, the NO donor sodium nitroprusside significantly enhanced the magnitude of the vagal-induced bradycardia, showing the downstream intracellular pathways activated by NO were intact. These results suggest that neuronal NO facilitates vagally induced bradycardia via a presynaptic modulation of neurotransmission.

Raised extracellular potassium attenuates the sympathetic modulation of sino-atrial node pacemaking in the isolated guinea-pig atria.

Intense exercise or myocardial ischaemia can significantly increase both the concentration of extracellular potassium ([K(+)](o)) and cardiac sympathetic nerve activity. Since changes in [K(+)](o) modulate membrane currents involved in sino-atrial node pacemaking, in particular the voltage-sensitive hyperpolarization-activated current (I(f)), we investigated whether raised [K(+)](o) (from 4 mM to 8 or 12 mM) could directly affect the heart rate response to cardiac sympathetic nerve stimulation (SNS). In the isolated guinea-pig atrial-right stellate ganglion preparation, raised [K(+)](o) significantly decreased the maximum diastolic potential, amplitude and maximum rate of rise of the upstroke of sino-atrial node pacemaker action potentials in 8 and 12 mM [K(+)](o) (P < 0.05). At 12 mM [K(+)](o) these effects were associated with significant decreases in baseline heart rate (4 mM [K(+)](o) = 187 +/- 5 beats min(-1) (bpm); 12 mM = 144 +/- 11 bpm; P < 0.05) and the heart rate response to SNS (1, 3 and 5 Hz; P < 0.05). A 10 % increase in the baseline heart rate with sympathetic activation (3 Hz) was associated with a significant enhancement of the slope of the pacemaker diastolic depolarization at 4 mM [K(+)](o) (increased by 16 +/- 6 %; n = 7; P < 0.05), but not with raised [K(+)](o). When the I(f) current was blocked with 2 mM caesium (n = 8), 12 mM [K(+)](o) had no effect on baseline heart rate and the heart rate response to 3 Hz SNS. The heart rate response to bath-applied noradrenaline (0.01-100 microM) was significantly attenuated by 12 mM [K(+)](o) (at 4 mM [K(+)](o,) EC(50) = -6.31 +/- 0.18; at 12 mM [K(+)](o,) EC(50) = -5.80 +/- 0.10; n = 6, ANOVA, P < 0.05). In conclusion, extreme physiological levels of [K(+)](o) attenuate the positive chronotropic response to cardiac sympathetic activation due to decreased activation of the I(f) current. This is consistent with raised [K(+)](o) protecting the myocardium from potentially adverse effects of excessive noradrenaline. Experimental Physiology (2001) 86.1, 19-25.

Do unusual site-specific population dynamics of rodent reservoirs provide clues to the natural history of hantaviruses?

Between January 1995 and November 1997, longitudinal mark-recapture studies of rodent hosts of hantaviruses in a disturbed microhabitat within a shortgrass prairie ecosystem in southeastern Colorado (USA) were conducted. The site was distinguished by edaphic and floristic characteristics unique to this area and associated with historical land use patterns, as well as the year-around availability of water from a functioning windmill. Populations of two common rodent species that are hosts for hantaviruses, Peromyscus maniculatus and Reithrodontomys megalotis, had unusually rapid turnover, a younger age structure, and a much lower prevalence of antibody to Sin Nombre virus than did populations at nearby sites in more typical shortgrass prairie and canyon habitats. Based on these findings, we suggest that a stable resident population of the reservoir is critical to the maintenance of hantaviruses at a given site, and we hypothesize that long-lived, persistently infected rodents are the principal transseasonal reservoir of hantaviruses.

Pregnancy and labor increase the capacity of human myometrial cells to secrete parathyroid hormone-related protein.

Parathyroid hormone-related protein (PTHrP), a oncofetal gene product possessing smooth muscle relaxant properties, has been found in rat and human uterine smooth muscle cells (USMC) where it is postulated to regulate myometrial tone and/or blood flow. Studies investigating the gestational regulation of PTHrP in human USMC have not been performed. This study was conducted to determine if pregnancy alters the capacity of USMC to secrete or respond to PTHrP. USMC cultures were established from 8 hysterectomy specimens (H) and 7 non-laboring (NP) and 5 laboring term pregnant uterine biopsies (LP). PTHrP secretion was measured at baseline and in response to TGF-beta1 using a immunoradiometric assay. The USMC response to PTHrP was assessed by incubating cultures with human (1-34)PTHrP and measuring cellular cAMP by radioimmunoassay. We found that cultures from the groups did not differ with respect to basal PTHrP secretion. TGF-beta1, on the other hand, produced dose-dependent increases in secreted PTHrP in each group such that LP>NP>H at 12 hrs and LP>NP and H 24 hrs. Maximal responses were found at 24 hrs in cells treated with 10 ng/ml TGF-beta1 (LP: 2034+/-366 vs NP: 1485+/-427; H: 1250+/-202 fmol/mg). Incubation of cultures with PTHrP produced dose-dependent increases in cAMP production, with 10(-7) M increasing levels by 64%. Neither pregnancy nor labor significantly affected the cAMP response. These findings indicate that the human myometrium has the capacity to increase PTHrP secretion during pregnancy and labor through a TGF-beta-dependent pathway. Such findings are consistent with a role of PTHrP in enhancing uterine blood flow.

Peripheral pre-synaptic pathway reduces the heart rate response to sympathetic activation following exercise training: role of NO.

OBJECTIVES: We tested the hypothesis that the attenuated heart rate (HR) response to sympathetic activation following swim training in the guinea pig (Cavia porcellus) results from a peripheral modulation of pacemaking by nitric oxide (NO). METHODS: Nitric oxide synthase (NOS) inhibition on the increase in heart rate with sympathetic nerve stimulation (SNS) was investigated in the isolated guinea pig double atrial/right stellate ganglion preparation from exercise trained (6-weeks swimming, n=20) and sedentary animals (n=20). Western blot analysis for neuronal nitric oxide synthase (nNOS) was performed on the stellate ganglion from both groups. RESULTS: Relative to the control group, the exercise group demonstrated typical exercise adaptations of increased ventricular weight/body weight ratio, enhanced skeletal muscle citrate synthase activity and higher concentrations of [3H]ouabain binding sites in both skeletal and cardiac tissue (P<0.05). The increase in heart rate (bpm) with SNS significantly decreased in the exercise group (n=16) compared to the sedentary group (n=16) from 30+/-5 to 17+/-3 bpm at 1 Hz; 67+/-7 to 47+/-4 bpm at 3 Hz; 85+/-9 to 63+/-4 bpm at 5 Hz and 101+/-9 to 78+/-5 bpm at 7 Hz stimulation (P<0.05). The increase in heart rate with cumulative doses (0.1-10 microM) or a single dose (0.1 microM) of bath-applied norepinephrine expressed as the effective doses at which the HR response was 50% of the maximum response (EC50) were similar in both exercise (EC50 -6.08+/-0.16 M, n=8) and sedentary groups (EC50 -6.18+/-0.07 M, n=7). Trained animals had significantly more nNOS protein in left stellate ganglion compared to the sedentary group. In the exercise group, the non-isoform selective NOS inhibitor, N-omega nitro-L-arginine (L-NA, 100 microM) caused a small but significant increase in the heart rate response to SNS. However, the positive chronotropic response to sympathetic nerve stimulation remained significantly attenuated in the exercise group compared to the sedentary group during NOS inhibition (P<0.05). CONCLUSIONS: Our results indicate that there is a significant peripheral pre-synaptic component reducing the HR response to sympathetic activation following training, although NO does not play a dominant role in this response.

Exercise training enhances relaxation of the isolated guinea-pig saphenous artery in response to acetylcholine.

The effects of exercise training were investigated on the vascular responses in the isolated guinea-pig saphenous artery. Exercising animals swam 5 days week-1 for 6 weeks (60 min day-1 for weeks 1 and 2; 75 min day-1 for weeks 3 and 4; 90 min day-1 for weeks 5 and 6), while control animals were placed into shallow water for the same duration. Trained animals had significantly higher ventricular:body weight ratios, increased citrate synthase activity in the latissimus dorsi, and enhanced Na+ pump concentrations in the latissimus dorsi and gastrocnemius muscles (P < 0.05). In vitro isometric techniques were used to measure constriction and relaxation responses of saphenous artery rings from trained and control animals. There were no significant differences in the constriction responses to KCl (50 mm) and phenylephrine (0.3-100 microM) in arterial rings from control versus trained animals. Relaxation responses to acetylcholine (10 microM; ACh-relaxation), following preconstriction with phenylephrine (10 microM), were significantly enhanced in rings from trained animals (P < 0.05). Acetylcholine relaxed the vessels to 47 +/- 6% (control) and 18 +/- 3% (trained) of the preconstriction responses to phenylephrine. The nitric oxide synthase inhibitor N G-nitro-L-arginine (L-NA; 50 microM) significantly attenuated the ACh-relaxation in control and trained animals (P < 0.05). The effect of L-NA on the ACh-relaxation was significantly larger in trained (change in ACh-relaxation with L-NA = 29 +/- 9%) than control (14 +/- 3%) animals (P < 0.05). In conclusion, exercise training enhanced the ACh-relaxation of the isolated guinea-pig saphenous artery. Inhibition of nitric oxide synthase attenuated the ACh-relaxation of rings from control and trained animals, but this effect was significantly larger in the vessels from trained animals. These results are consistent with the idea that nitric oxide could contribute to the enhanced ACh-relaxation of the saphenous artery with exercise training.

Androgen receptor messenger ribonucleic acid in brains and pituitaries of male rhesus monkeys: studies on distribution, hormonal control, and relationship to luteinizing hormone secretion.

Because the distribution and hormonal regulation of the androgen receptor (AR) mRNA in brains and pituitaries of adult rhesus monkeys have not been studied, we cloned and sequenced a 329-base pair segment of the 5' coding region of the rhesus AR cDNA. Monkey AR cDNA was 99% identical with the human sequence and 96% homologous with the rat sequence. Using a ribonuclease protection assay, we studied the distribution and regulation of AR mRNA in brains and anterior pituitary glands of three groups of male rhesus monkeys: intact (n = 3), castrated (Cx, n = 4), and Cx treated with testosterone (n = 6). Serum testosterone levels of Cx males treated with testosterone differed significantly (p < 0.05) in the morning but not in the evening hours from those in intact controls. Serum LH concentrations were significantly suppressed (p < 0.05) in both morning and evening serum samples of testosterone-treated males compared to intact controls. We found the highest concentrations of AR mRNA in the medial basal hypothalamus, the bed nucleus of the stria terminalis, the medial preoptic area-anterior hypothalamus, and the lateral dorsomedial hypothalamus. Intermediate amounts were found in the septum and amygdala. Low amounts were found in the hippocampus, cingulate cortex, parietal cortex, and cerebellum. The anterior pituitary gland also contained a large amount of AR mRNA. Surprisingly, neither Cx for 3 wk nor Cx plus testosterone replacement for 3 wk significantly affected AR mRNA in any brain area or in the pituitary gland. The present study demonstrates that the effectiveness of testosterone as a regulator of LH secretion in male monkeys is not related to changes of AR mRNA in the brain or pituitary gland. It appears that AR mRNA in the monkey brain and pituitary gland is not regulated at the transcriptional level by androgen.

Nitric oxide inhibits the positive chronotropic and inotropic responses to sympathetic nerve stimulation in the isolated guinea-pig atria.

This study was designed to determine whether nitric oxide (NO) modulates the positive chronotropic and inotropic (in paced atria) responses to cardiac sympathetic nerve stimulation (SNS) in the isolated guinea-pig double atrial/right stellate ganglion preparation. The ganglion was stimulated at 1, 2, 3 and 5 Hz at constant voltage and the changes in heart rate or force of contraction were measured. The selective neuronal NO synthase (nNOS) inhibitors TRIM (1-(2-trifluoromethylphenyl) imidazole; 100 microM) and 7-NiNa (Na+ salt of 7-nitroindazole; 100 microM) significantly enhanced the positive chronotropic and inotropic responses to SNS. Similar results for heart rate were seen with the non-isoform-selective NOS inhibitor N(omega)nitro-L-arginine (L-NA; 100 microM). All effects were reversed with L-arginine (1 mM). The NO donor sodium nitroprusside (SNP; 100 microM) increased baseline heart rate and force of contraction, and attenuated the positive chronotropic and inotropic responses to SNS. SNP also decreased the positive chronotropic response to bath-applied noradrenaline (NA; 1 microM). In contrast, 7-NiNa did not alter the increase in heart rate with bath-applied NA (0.1 or 1 microM). The guanylyl cyclase inhibitor ODQ (10 microM) enhanced (mimicking nNOS inhibition) and the cyclic GMP (guanosine 3':5'-cyclic monophosphate) analogue 8-Br-cGMP (8-bromoguanosine 3':5'-cyclic monophosphate; 1 mM) attenuated (mimicking exogenous NO) the positive inotropic response to SNS. Taken together, these results are consistent with endogenous NO, synthesized from nNOS, inhibiting the positive chronotropic and inotropic responses evoked by cardiac SNS via a cyclic GMP-dependent pathway.

NO-cGMP pathway accentuates the decrease in heart rate caused by cardiac vagal nerve stimulation.

The role of the cardiac muscarinic-receptor-coupled nitric oxide (NO) pathway in the cholinergic control of heart rate (HR) is controversial. We investigated whether adding excessive NO or its intracellular messenger cGMP could significantly modulate the HR response to vagal nerve stimulation (VNS) in the anesthetized rabbit and isolated guinea pig atria. The NO donor molsidomine (0.2 mg/kg iv) significantly enhanced the decrease in HR seen with right VNS (5 Hz, 5 V, 30 s) in vivo. A qualitatively similar effect was seen with the NO donor sodium nitroprusside (SNP; 10 and 100 microM) during VNS in vitro. This effect was still present when the baseline shift in HR caused by SNP was eliminated by using the specific hyperpolarization-activated current antagonist 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)-pyrimidinium chloride (ZD-7288, 1 microM). The accentuated decrease in HR with SNP during VNS was mimicked by the stable analog of cyclic GMP, 8-bromoguanosine 3',5'-cyclic monophosphate (0.5 mM). This, however, was not seen with bath application of the stable analog of acetylcholine, carbamylcholine chloride (100 nM). We conclude that excessive NO enhances the magnitude of the decrease in HR caused by VNS. This effect appears to involve a presynaptic action via a cGMP-dependent pathway because it was not mimicked by bath-applied carbamylcholine chloride.

Effect of nitric oxide synthase inhibition on the sympatho-vagal contol of heart rate.

The role of nitric oxide (NO) in the sympatho-vagal control of heart rate was investigated in the cardiac sympathectomized and vagotomized anaesthetised rabbit and in the isolated guinea-pig atria with intact vagus nerve. Specific inhibition of neuronal nitric oxide synthase (nNOS) with 1-(2-trimethylphenyl) imidazole (TRIM, 50 mg kg(-1) i.v. in vivo) significantly enhanced the magnitude of the change in heart rate (HR) with sympathetic nerve stimulation (SNS, 31.6+/-4.5 bpm control vs. 49.7+/-6.0 bpm in TRIM, P < 0.05, 10 Hz). This effect was reversed by L-arginine (deltaHR 37.2+/-4.1 bpm, 50 mg kg(-1) i.v.). An enhanced HR response to SNS was also seen with the non-isoform specific inhibitor, N-omega-nitro-L-arginine (L-NA, 50 mg kg(-1) i.v.). Infusing isoprenaline (0.2 microg kg(-1) min(-1)) did not mimic the change in HR response to SNS with TRIM. There was, however, no significant effect of inhibition of NOS with TRIM L-NA or NG-monomethyl-L-arginine (L-NMMA, 20 mg kg(-1) i.v.) on the magnitude of the change in HR with vagal nerve stimulation (5 Hz) in vivo. There was also no significant effect of NOS inhibition on the change in HR with vagal nerve stimulation in vivo in the presence of pre-adrenergic stimulation or in the presence of propranolol (0.5 mg kg(-1) i.v., 2, 5 and 10 Hz stimulation). This result was confirmed in the isolated guinea-pig atria with the specific nNOS inhibitor, 7-nitroindazole (7-NiNa, 100 microM) at 1, 2, 3 or 5 Hz stimulation frequency. Our data suggest that endogenous NO plays an inhibitory role in cardiac sympathetic neurotransmission, but there was no convincing evidence from our results for a major role for endogenous NO in vagal control of heart rate, with or without prior adrenergic stimulation.

Immunocytochemical localization of androgen receptors in brains of developing and adult male rhesus monkeys.

We localized immunoreactive androgen receptors in the central nervous system (CNS) of fetal and adult male rhesus macaques by immunocytochemisty using an affinity-purified polyclonal antibody to the first 21 amino acids of the human androgen receptor (AR). This antibody caused a shift in the mobility of AR-bound 3H-DHT on a sucrose gradient and recognized a protein of approx 116 kDa on Western blot. Other criteria for specificity are presented. We localized AR in the diencephalon of male rhesus monkey fetuses. Immunoreactive neurons were found in the medial hypothalamic area and the ventromedial nucleus of the hypothalamus on days 47, 61, and 124 of gestation. At 124 d of gestation, AR immunoreactivity was aslo found in the arcuate nucleus. AR immunostaining was not found in other diencephalic structures in fetal life, including the preoptic area. In the adult monkey, neurons in ventromedial, dorsomedial, and arcuate nuclei of the hypothalamus; cortical, medial, and accessory basal nuclei of the amygdala; and regions of the hippocampus and the anterior pituitary gland contained immunoreactive AR. These data indicate that AR is found in specific areas of the CNS early in fetal development, but they also appear in other brain areas as the fetus grows. At 124 d of gestation (term, 167 d), the hypothalamic location of immunoreactive AR is similar to the adult.

Inhibition of nitric oxide synthase slows heart rate recovery from cholinergic activation.

The role of nitric oxide (NO) in the cholinergic regulation of heart rate (HR) recovery from an aspect of simulated exercise was investigated in atria isolated from guinea pig to test the hypothesis that NO may be involved in the cholinergic antagonism of the positive chronotropic response to adrenergic stimulation. Inhibition of NO synthesis with NG-monomethyl-L-arginine (L-NMMA, 100 micro M) significantly slowed the time course of the reduction in HR without affecting the magnitude of the response elicited by bath-applied ACh (100 nM) or vagal nerve stimulation (2 Hz). The half-times (t1/2) of responses were 3.99 +/- 0.41 s in control vs. 7. 49 +/- 0.68 s in L-NMMA (P < 0.05). This was dependent on prior adrenergic stimulation (norepinephrine, 1 micro M). The effect of L-NMMA was reversed by L-arginine (1 mM; t1/2 4.62 +/- 0.39 s). The calcium-channel antagonist nifedipine (0.2 micro M) also slowed the kinetics of the reduction in HR caused by vagal nerve stimulation. However, the t1/2 for the reduction in HR with antagonists (2 mM Cs+ and 1 micro M ZD-7288) of the hyperpolarization-activated current were significantly faster compared with control. There was no additional effect of L-NMMA or L-NMMA+L-arginine on vagal stimulation in groups treated with nifedipine, Cs+, or ZD-7288. We conclude that NO contributes to the cholinergic antagonism of the positive cardiac chronotropic effects of adrenergic stimulation by accelerating the HR response to vagal stimulation. This may involve an interplay between two pacemaking currents (L-type calcium channel current and hyperpolarization-activated current). Whether NO modulates the vagal control of HR recovery from actual exercise remains to be determined.

Distribution of aromatase cytochrome P450 messenger ribonucleic acid in adult rhesus monkey brains.

The conversion of androgens to estrogens by aromatase cytochrome P450 (P450arom) is an important step in the mechanism of androgen action in the brain. However, the distribution of P450arom mRNA in adult rhesus monkey brains has not been studied because specific probes have not been available. To address this deficit, we cloned and sequenced a 455-basepair segment of the 5' coding region of the rhesus P450arom cDNA. Total RNA was extracted from a rhesus monkey placenta (Day 47 of gestation and subjected to reverse transcriptase (RT) polymerase chain reaction (PCR) using consensus oligonucleotide primers selected from published human and rat P450arom DNA sequences. The RT-PCR product was subcloned into a vector and sequenced. The monkey P450arom cDNA was 97% identical to the human sequence but shared only 86% homology with the rat sequence. We then developed a ribonuclease protection assay using a monkey P450arom cDNA and studied the distribution of P450arom mRNA in adult monkey brains. This assay protected two RNA fragments, one 455 nucleotides (nt) in length and the second approximately 300 nt. The relative distribution of P450arom mRNA (the 455-nt fragment) between brain areas of the adult (n = 3) was high in the bed nucleus of the stria terminalis > medial preoptic/anterior hypothalamus > amygdala; intermediate in the medial basal hypothalamus (infundibular nucleus, median eminence, ventromedial nucleus) > lateral preoptic/anterior hypothalamus; and low in the septum > lateral-dorsal-medial hypothalamus. P450arom mRNA was undetectable in cingulate and parietal cortex, hippocampus, and cerebellum. P450arom activity, as measured by the 3H2O assay, correlated well with the distribution of P450arom mRNA (the 455-nt protected fragment; r = 0.9) in the same tissues. A shorter protected RNA fragment was found in the medial basal hypothalamus, the bed nucleus of the stria terminalis, the amygdala, and the cingulate and parietal cortex but not in the other brain areas investigated. Its presence did not correlate with aromatase activity in brain tissue. This study describes the development of a ribonuclease protection assay using a monkey cDNA produced by RT-PCR and its usefulness for studying the distribution of P450arom mRNA in brains of nonhuman primates.