Vasorelaxation responses to insulin in laminar vessel rings from healthy, lean horses.

Hyperinsulinemia causes laminitis experimentally and is a risk factor for naturally occurring laminitis. The aim of this study was to investigate the effects of insulin on laminar vascular relaxation and to induce insulin-associated vascular dysfunction in vitro. Relaxation responses of isolated laminar arterial and venous rings to acetylcholine and insulin were evaluated. To alter vascular function in response to insulin, all vessel rings were incubated with insulin or vehicle, submaximally contracted, administered insulin again and relaxation responses recorded. Laminar arteries were also incubated with the mitogen-activated protein kinase (MAPK) inhibitor, PD-98059. Relaxation in response to acetylcholine was not different between arteries and veins, but veins relaxed less in response to insulin than arteries. In arteries incubated with insulin, the subsequent relaxation response to insulin was blunted. Veins had minimal relaxation to insulin regardless of incubation. Arteries incubated with PD-98059 relaxed more in response to insulin than arteries not exposed to PD-98059, indicating that MAPK plays a role in maintenance of basal tone in laminar arteries. A differing response of laminar veins and arteries to insulin-induced relaxation may be important in understanding the link between hyperinsulinemia and laminitis. In vitro induction of vascular dysfunction in response to insulin in laminar arteries may be useful for testing therapeutic interventions and for understanding the pathophysiology of laminitis.

Lymphocyte enzymatic antioxidant responses to oxidative stress following high-intensity interval exercise.

The purposes of this study were to 1) examine the immune and oxidative stress responses following high-intensity interval training (HIIT); 2) determine changes in antioxidant enzyme gene expression and enzyme activity in lymphocytes following HIIT; and 3) assess pre-HIIT, 3-h post-HIIT, and 24-h post-HIIT lymphocyte cell viability following hydrogen peroxide exposure in vitro. Eight recreationally active males completed three identical HIIT protocols. Blood samples were obtained at preexercise, immediately postexercise, 3 h postexercise, and 24 h postexercise. Total number of circulating leukocytes, lymphocytes, and neutrophils, as well as lymphocyte antioxidant enzyme activities, gene expression, cell viability (CV), and plasma thiobarbituric acid-reactive substance (TBARS) levels, were measured. Analytes were compared using a three (day) × four (time) ANOVA with repeated measures on both day and time. The a priori significance level for all analyses was P < 0.05. Significant increases in superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) activities were observed in lymphocytes following HIIT. No significant increases in lymphocyte SOD, CAT, or GPX gene expression were found. A significant increase in TBARS was found immediately post-HIIT on days 1 and 2. Lymphocyte CV in vitro significantly increased on days 2 and 3 compared with day 1. Additionally, there was a significant decrease in CV at 3 h compared with pre- and 24 h postexercise. These findings indicate lymphocytes respond to oxidative stress by increasing antioxidant enzyme activity. Additionally, HIIT causes oxidative stress but did not induce a significant postexercise lymphocytopenia. Analyses in vitro suggest that lymphocytes may become more resistant to subsequent episodes of oxidative stress. Furthermore, the analysis in vitro confirms that lymphocytes are more vulnerable to cytotoxic molecules during recovery from exercise.

Expression of vascular endothelial growth factor in canine oral malignant melanoma.

Serum, plasma and tissue expression of vascular endothelial growth factor (VEGF) was measured in 20 dogs previously diagnosed histologically with oral melanoma. The concentrations of VEGF in serum and plasma were significantly higher in dogs with melanoma than in a control population (P

Pharmacologic identification of putative D1 dopamine receptors in feline kidneys.

The presence of dopamine (DA) receptors in feline kidneys is a matter of contention. Radioligand binding and Western blotting studies were employed to determine whether DA receptors are present in feline kidneys. The pharmacologic profile of the selective D1-receptor antagonist [3H]-SCH 23390 was studied in renal cortical membrane preparations from cats by conducting saturation binding isotherm and competitive binding experiments. [3H]-SCH 23390 bound to feline renal cortical membranes in a manner consistent with labeling of a D1-like receptor. The binding profile revealed a single site D1-like or D1 receptor in the feline renal cortex with a Kd = 7.8 +/- 1.0 nmol/L and Bmax = 76.5 +/- 19.5 fmol/mg. Competitive binding studies for [3H]-SCH 23390 against unlabeled agonists yielded the following Ki values and rank order of competition: SKF38393 (Ki = 0.47 +/- 0.26 micro m) > fenoldopam (Ki = 3.12 +/- 1.1 micro m) > DA (Ki = 933.1 +/- 1.6 micro m). Competitive binding studies for [3H]-SCH-23390 against unlabeled antagonists yielded the following rank order of competition: SCH 23390 (Ki = 1.97 +/- 0.81 micro m) > spiperone (Ki = 3.79 +/- 0.79) > metoclopramide (Ki = 4.26 +/- 2.4 micro m). Western blot analysis with anti-DA D1 receptor antibodies detected a single band with Mr of 74 kDa corresponding to a D1 DA receptor. These results suggest that a putative D1-like or D1 receptor exists in feline kidneys different from those previously identified in rat, dog or human kidneys.

Estradiol plus progesterone treatment modulates select elements of the proinflammatory cytokine cascade in steers: attenuated nitric oxide and thromboxane B2 production in endotoxemia.

Estradiol plus progesterone (EP) implants have been shown to favorably alter the time course or decrease the severity of many of the clinical manifestations associated with coccidiosis and endotoxemia in calves. This study evaluated the effect of EP treatment on plasma tumor necrosis factor-alpha (TNF), thromboxane (TXB), prostacyclin (PRC), nitrite and nitrate (NO[x]), and cortisol. Holstein steer calves were divided into four groups: control, EP, endotoxin (LPS), and EP + LPS (n = five/group). Estradiol/progesterone pellets (Synovex-S) were implanted subcutaneously when calves reached 20 wk of age. One week after implantation, calves were injected i.v. with endotoxin (i.e., lipopolysaccharide; LPS, 0.6 microgram/kg of BW) or nonpyrogenic saline placebo. Body temperature was measured and blood was collected before injection and at 1, 2, 3, 4, 6, and 8 h thereafter. Plasma concentrations of TNF, cortisol, TXB, PRC, NO[x], were measured. Body temperature increased in both LPS and LPS-EP calves, but had returned to normal by 6 h in the LPS-EP group (P < 0.05). Plasma TNF and cortisol increased after LPS (P < 0.01), but were not differentially affected by EP treatment. Likewise, EP did not affect the magnitude of increase in LPS-induced PRC, but EP decreased the magnitude of increase in TXB (P < 0.05). Plasma NO[x]) levels were increased (P < 0.01) in calves after LPS; treatment with EP attenuated the LPS-associated increase in plasma NO[x] levels. These results suggest that EP exerts specific effects on different components of the proinflammatory cytokine cascade. Although the initiation of responses mediated by TNF, cortisol, and PRC do not seem to be differentially affected by EP, components of the nitric oxide- and TXB-axis responses to LPS are decreased in calves pretreated with EP.

Reduced growth of calves and its reversal by use of anabolic agents.

Disease has profound effects on the immune system, endocrine system, and on the growth process. Since diseases are catabolic to the animal, there is current interest in the possible role of anabolic hormones to counter the effects of disease in general and minimize the effects of a disease process on growth and development. A number of anabolic hormones, such as growth hormone (GH) and estradiol + progesterone (EP), have been studied for their role in enhancing growth and stimulating immune function and are thus candidates for hormonal intervention in disease processes. GH has been shown to be effective in countering some of the deleterious effects of endotoxemia but was ineffective in a parasitic disease model. Studies with EP have shown similar success with both endotoxemia and a parasitic disease model. Moreover, GH and EP do not share a common mechanism of action, suggesting that the effects are not simply due to anabolic actions. While the mechanism of action of GH in endotoxemia has been examined, the effects of EP are via an unknown mechanism, possibly by inhibition of IL-I action or inhibition of nitric oxide overproduction.

Molecular and pharmacological characterization of the canine brainstem alpha-2A adrenergic receptor.

This study characterizes the alpha2-adrenergic receptors present in canine brainstem. Radioligand binding and reverse transcriptase-polymerase chain reaction (RT-PCR) experiments were performed in canine brainstem to identify the receptors present and determine the pharmacological properties of these receptors. The pKi values derived from radioligand competition curves for a number of adrenergic receptor agents at the four alpha2-adrenergic receptor subtypes were compared to the canine brainstem. The pKi values at the canine brainstem alpha2-adrenergic receptor were consistent with the presence of the alpha2A-adrenergic receptor. To determine whether the canine brainstem expressed the message for the alpha2A-adrenergic receptor, RT-PCR was performed with specific primers for the four subtypes of alpha2-adrenergic receptors. In the canine brainstem, only the primers corresponding to a region in the human alpha2A-adrenergic receptor produced a PCR product. No bands were detected in the canine brainstem lanes with the alpha2B-, alpha2C-, or alpha2D-receptor primers. These data suggest that the canine brainstem contains the alpha2A-adrenergic receptor.

Selectivity of atipamezole, yohimbine and tolazoline for alpha-2 adrenergic receptor subtypes: implications for clinical reversal of alpha-2 adrenergic receptor mediated sedation in sheep.

The alpha2-adrenergic receptor antagonists, yohimbine, atipamezole and tolazoline, are used in veterinary medicine as reversal agents for the sedative/hypnotic effects of alpha2-agonists. Ruminants have increased sensitivity to the sedative/hypnotic effects of alpha2-agonists compared to other species. The receptors mediating the sedative effects of alpha2-agonists are located primarily on locus coeruleus neurons in the pons of the lower brainstem. Four pharmacological subtypes of the alpha2-adrenergic receptor (A,B, C and D) have been identified based on differences in ligand affinity. The aim of this study was to: 1) determine the pharmacological profile of atipamezole, yohimbine and tolazoline at the four alpha2-adrenergic receptor subtypes and; 2) determine whether these agents differ in their affinities at the alpha2-adrenergic receptor present in the sheep brainstem. In inhibition binding studies against the selective alpha2-adrenergic receptor ligand [3H]-MK-912, tolazoline showed the lowest affinity for all four alpha2-adrenergic receptor subtypes compared to yohimbine and atipamezole. The affinities of yohimbine and atipamezole were similar at the alpha2A-, alpha2B- and alpha2C-adrenergic receptors but differed by approximately 100 fold at the alpha2D-adrenergic receptor. Atipamezole had a 100 fold higher affinity at the alpha2D-adrenergic receptor when compared to yohimbine. To determine the ligand binding characteristics of these agents at the alpha2-adrenergic receptor in sheep brainstem, membranes were labelled with [3H]-MK-912 and inhibition competition curves were performed. Atipamezole showed approximately a 100 fold higher affinity for the sheep brainstem alpha2-adrenergic receptor compared to yohimbine which was similar to what was observed for the alpha2D-adrenergic receptor in PC12 cells transfected with RG-20. The results from these studies suggest that atipamezole has a high affinity for the alpha2D-adrenergic receptor that appears to be the receptor subtype in sheep brainstem.

ANG II-related myocardial damage: role of cardiac sympathetic catecholamines and beta-receptor regulation.

The objectives of this study were 1) to determine whether ANG II-induced myocardial damage (ANG Dam) is mediated via the beta1-adrenergic receptor, 2) to elucidate whether adrenal medulla or cardiac sympathetic neuron catecholamines are responsible for ANG Dam, and 3) to determine whether the lack of damage after 3 days of elevated ANG II levels is due to beta1-receptor downregulation. To this end, ANG II was administered to rats 1) that were treated with a beta-receptor blocker, 2) after adrenal medullectomy and/or cardiac sympathectomy, and 3) for 3 or 8 days. ANG II caused both myocyte necrosis and coronary vascular damage after adrenal medullectomy but not after cardiac sympathectomy. There was a 38 and 55% decrease in beta-receptor density after 3 and 8 days, respectively, of ANG II infusion, and an upregulation to control levels 5 days after a 3-day ANG II infusion was stopped. We conclude that cardiac sympathetic neuron catecholamines are responsible for ANG Dam and that the acute nature of this damage is associated with a downregulation of beta1-adrenergic receptors.

Affinity of detomidine, medetomidine and xylazine for alpha-2 adrenergic receptor subtypes.

alpha 2-Adrenergic receptor agonists are widely used in veterinary medicine as sedative/hypnotic agents. Four pharmacological subtypes of the alpha 2-adrenergic receptor (A, B, C and D) have been identified based primarily on differences in affinity for several drugs. The purpose of this study was to examine the affinities of the sedative agents, xylazine, detomidine and medetomidine at the four alpha 2-adrenergic receptor subtypes. Saturation and inhibition binding curves were performed in membranes of tissues containing only one subtype of alpha 2-adrenergic receptor. The KD for the alpha 2-adrenergic receptor radioligand, [3H]-MK-912, in HT29 cells (alpha 2A-), neonatal rat lung (alpha 2B-), OK cells (alpha 2C-) and PC12 cells transfected with RG20 (alpha 2D-) were 0.38 +/- 0.08 nM, 0.70 +/- 0.5 nM. 0.07 +/- 0.02 nM and 0.87 +/- 0.03 nM, respectively. Detomidine and medetomidine had approximately a 100 fold higher affinity for all the alpha 2-adrenergic receptors compared to xylazine but neither agonist displayed selectivity for the alpha 2-adrenergic receptor subtypes. These data suggest that available sedative/hypnotic alpha 2-adrenergic receptor agonists can not discriminate between the four known alpha 2-adrenergic receptor subtypes.

Activation of alpha-2 adrenergic receptors inhibits norepinephrine release by a pertussis toxin-insensitive pathway independent of changes in cytosolic calcium in cultured rat sympathetic neurons.

Whole-cell electrophysiological studies suggest that sympathetic nerve alpha-2 adrenergic receptors are coupled to voltage-dependent N-type calcium channels through the Gi family of proteins to inhibit neurotransmitter release. Because most nerve terminals are too small for direct electrophysiological recordings, the aim of this study was to examine the relationship between alpha-2 adrenergic receptor-mediated inhibition of norepinephrine release and the rise in cytosolic calcium in neurites from cultured sympathetic neurons. In cultured rat superior cervical ganglion neurons, the alpha-2 adrenergic receptor agonists, UK-14304 (0.01-10 microM) and oxymetazoline (0.1-10 microM), and the N-type calcium channel blocker, omega-conotoxin GVIA (0.1-10 nM), inhibited the release of tritiated norepinephrine in response to electrical stimulation (1 Hz, 30 pulses, 0.1 ms, 70 V). The inhibitory effect of the alpha-2 adrenergic receptor agonists was not altered by pretreatment with pertussis toxin (200 ng/ml, 18 h), although pertussis toxin blocked the inhibition of forskolin-stimulated cAMP accumulation by UK-14304. In fura-2 loaded cells, electrical stimulation (1 Hz, 30 pulses, 0.1 ms, 70 V) increased cytosolic calcium in sympathetic neuronal processes. Blockade of N-type calcium channels with omega-conotoxin (1 and 10 nM) reduced the rise in cytosolic calcium by 25 +/- 3% and 52 +/- 6%, respectively, whereas UK-14304 and oxymetazoline did not alter the electrically stimulated rise in cytosolic calcium. These data suggest that blockade of N-type calcium channels with omega-conotoxin GVIA inhibits stimulated norepinephrine release and cytosolic calcium measured with fura-2 at similar concentrations, whereas activation of alpha-2 adrenergic receptor inhibits norepinephrine release by a pathway that is insensitive to pertussis toxin and changes in cytosolic calcium in neurites from cultured rat superior cervical ganglion cells.

Activation of protein kinase C by angiotensin II decreases beta 1-adrenergic receptor responsiveness in the rat heart.

Cardiac beta-adrenergic receptors are the primary driving force for the enhancement of contractility in response to sympathetic stimulation. Angiotensin II influences cardiac function by modulating sympathetic activity and by activating cardiac angiotensin II receptors. The aim of this study was to determine whether activation of cardiac angiotensin II receptors modulates the responsiveness of the heart to beta-adrenergic receptor activation. Male Sprague-Dawley rats were anesthetized and the hearts isolated and perfused with oxygenated Krebs-Henseleit buffer (KHB). Coronary artery perfusion pressure, left ventricular pressure (LVP), left ventricular dP/dtmax, and heart rate (HR) were measured. Bolus administration of the beta-adrenergic receptor agonists, isoproterenol, dobutamine, and salbutamol, produced dose-related increases in LVP, LV dP/dt(max), and HR. Addition of angiotensin-II (10-100 nM) to the KHB slightly increased coronary perfusion pressure but did not alter baseline LVP, LV dP/dt(max), or HR. Angiotensin II reduced the increase in LVP, LV dP/dt(max), and HR elicited by isoproterenol and dobutamine but did not affect responses to salbutamol. The inhibitory effect of angiotensin II was blocked by the AT1-receptor antagonist, losartan, and the protein kinase C inhibitor, calphostin C (50 nM). Activation of protein kinase C with phorbol-12, 13-dibutyrate (PDBu; 10 nM) reduced cardiac responses to all three agonists, although the effects were less on responses elicited by salbutamol. These data suggest that activation of protein kinase C by angiotensin II decreases the responsiveness of the rat heart to beta 1-adrenergic stimulation and that angiotensin II-mediated protein kinase C activation may differ from that activated by phorbol esters.

Neuropeptide Y inhibits adrenergic transmitter release in cultured rat superior cervical ganglion cells by restricting the availability of calcium through a pertussis toxin-sensitive mechanism.

Neuropeptide Y has been reported to inhibit the release of the adrenergic transmitter from sympathetic nerves in many tissues. The purpose of this study was to determine the mechanism of the inhibitory effect of neuropeptide Y on the release of the adrenergic transmitter in cultured superior cervical ganglion cells prelabeled with tritiated norepinephrine. In cultured superior cervical ganglion cells superfused with a HEPES-buffered saline, electrical field stimulation (1 Hz, 30 pulses, 1 ms, 60 V) increased the fractional overflow of tritium. Neuropeptide Y (50 nM) attenuated this depolarization-induced increase in transmitter release. The nonhydrolyzable cAMP analog, 8-(4-chlorophenylthio)cyclic AMP (100 microM) and the potassium channel blockers, tetraethylammonium chloride (1 mM) and 4-aminopyridine (300 microM) potentiated the electrically stimulated increase in fractional tritium overflow but failed to alter the inhibitory effect of neuropeptide Y on fractional tritium overflow. Increasing the calcium concentration in the superfusion fluid from 1.8 to 5.4 mM potentiated the electrically stimulated increase in fractional tritium overflow and attenuated the inhibitory effect of neuropeptide Y. Reduction of superfusion fluid calcium concentration to 0.5 mM decreased electrically stimulated fractional tritium overflow and augmented the inhibitory effect of NPY on release of tritium. The fractional release of tritium in response to the calcium ionophore, ionomycin, was not significantly altered by neuropeptide Y. In Fura-2-loaded isolated sympathetic neurites obtained from superior cervical ganglia explants, the depolarization-induced (54 mM KCl) increase in cytosolic calcium was attenuated by neuropeptide Y (50 nM).(ABSTRACT TRUNCATED AT 250 WORDS)

Contribution of calcium to isoproterenol-stimulated lipolysis in the isolated perfused rabbit heart.

The present study was performed to investigate the contribution of adenosine 3',5'-cyclic monophosphate (cAMP) and calcium to isoproterenol-stimulated lipolysis in the isolated rabbit heart perfused with Krebs-Henseleit buffer according to the method of Langendorff. Isoproterenol (0.05-1.5 nmol) increased glycerol output, left ventricular dP/dtmax, and heart rate but decreased coronary perfusion pressure. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (50 microM) failed to alter the basal or isoproterenol-induced increase in glycerol output, whereas cilostamide (5 microM) enhanced basal and inhibited isoproterenol-stimulated glycerol output. Inhibition of adenylyl cyclase with (phenylisopropyl)adenosine reduced isoproterenol-stimulated mechanical parameters but had no effect on basal or isoproterenol-stimulated glycerol output, whereas the cAMP analogue 8-(4-chlorophenylthio)-cAMP did not increase glycerol output but produced changes in mechanical parameters similar to isoproterenol. Decreasing perfusion fluid calcium concentration from 1.2 to 0.5 mM or infusion of the calcium channel antagonist diltiazem (23 microM) abolished the increase in glycerol output in response to isoproterenol. Activation of adenylyl cyclase with forskolin increased glycerol output, but the increase was abolished by reducing perfusion fluid calcium concentration or by diltiazem. These data suggest that, in the rabbit heart, isoproterenol-stimulated lipolysis appears to be mediated predominantly by calcium as a secondary metabolic response provided by the increase in mechanical activity.

Cyclic AMP modulates but does not mediate the inhibition of [3H]norepinephrine release by activation of alpha-2 adrenergic receptors in cultured rat ganglion cells.

The purpose of this study was to determine whether a decrease in cyclic AMP accumulation mediates the inhibition of norepinephrine release in response to alpha-2 adrenergic receptor activation in cultured rat superior cervical ganglion cells. Superior cervical ganglia from neonatal rats were dissociated and cultured on collagen-coated plastic strips. Neurotransmitter release was assessed by measuring the fractional overflow of tritium in superfused cells prelabeled with [3H]norepinephrine. Intracellular cyclic AMP accumulation was measured using radioimmunoassay. Electrical field stimulation at 1 Hz, 30 pulses, 1 ms duration at 20 min intervals produced an increase in the fractional overflow of tritium that was composed predominantly of intact [3H]norepinephrine. The alpha-2 adrenergic receptor agonist UK-14,304 dose-dependently attenuated the increase in fractional tritium overflow elicited by electrical field stimulation. The adenylyl cyclase activator, forskolin, increased cyclic AMP accumulation in superior cervical ganglion cells and UK-14,304 dose-dependently inhibited forskolin-stimulated cyclic AMP accumulation. UK-14,304 had no effect on basal cyclic AMP accumulation or cyclic AMP accumulation during electrical field stimulation. Forskolin (1-10 microM) or the non-hydrolysable cAMP analog, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (1-100 microM), slightly increased basal and dose-dependently potentiated the increase in fractional tritium overflow in response to electrical stimulation. Despite enhancement by forskolin and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate of fractional tritium overflow caused by electrical field stimulation, UK-14304 (1-10 microM) reduced release to a similar degree as that observed in the absence of forskolin or 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of prejunctional alpha-2 adrenergic receptors involved in modulation of adrenergic transmitter release in the isolated perfused rat kidney.

The subclassification of alpha-2 adrenergic receptors into A and B subtypes is based on radioligand binding and functional studies. Radioligand binding studies also have suggested the existence of C and D subtypes, which have only been described as binding sites. This study was designed to determine the subtype of prejunctional alpha-2 adrenergic receptor involved in inhibition of norepinephrine release from sympathetic nerves in the rat kidney. Electrically induced (0.25 Hz, 50 V, 0.5 msec, 10 pulses) fractional tritium overflow was measured in kidneys isolated from male Sprague-Dawley rats and prelabeled with [3H]norepinephrine. The alpha-2 receptor antagonists rauwolscine and yohimbine did not enhance fractional tritium overflow, suggesting the lack of autoinhibition at this frequency of stimulation. The alpha-2 receptor agonist, UK-14,304, inhibited electrically stimulated fractional tritium overflow with an ED50 of 4.85 +/- 0.35 nM. pA2 values for various alpha receptor antagonists against UK-14,304-induced inhibition of fractional tritium overflow were: rauwolscine, 8.8; yohimbine, 8.1; prazosin, 7.4; BAM 1303, 7.5; SKF 104078, 6.4; phentolamine, 8.7; WB 4101, 8.1; corynanthine, 6.1; and ARC-239, 7.2. The correlation coefficients and slopes of the regression lines between pA2 values of alpha receptor antagonists at the prejunctional alpha-2 adrenergic receptor and the reported pKi values obtained from radioligand binding studies were: 0.48 and 0.82; 0.61 and 0.58; 0.53 and 0.85; and 0.89 and 1.05 for the alpha-2A, B, C and D adrenergic receptors, respectively. These data suggest that the prejunctional alpha-2 adrenergic receptor modulating [3H]norepinephrine release in the rat kidney most closely resembles the alpha-2D adrenergic receptor characterized by radioligand binding studies.

Mechanism of neurotransmitter release elicited by the preferential alpha 1-adrenergic receptor agonist phenylephrine in superfused superior cervical ganglion cells in culture.

Phenylephrine increased [3H]norepinephrine efflux and accumulation of cyclic AMP in cultured rat superior cervical ganglion cells superfused with Tyrode's solution. The purpose of this study was to determine the mechanism and relationship between these two events. Electrical stimulation (1-2 Hz), potassium chloride (50 mM), and the preferential alpha 1-adrenergic receptor agonist phenylephrine (1-100 microM) increased fractional tritium efflux, whereas methoxamine, cirazoline, and amidephrine were relatively ineffective. Phenylephrine, but not methoxamine and cirazoline, also increased cyclic AMP accumulation. Phenylephrine-induced tritium efflux was not altered by alpha- and beta-adrenergic receptor antagonists or by removal of extracellular calcium. Phenylephrine-induced cyclic AMP accumulation was blocked by the beta-adrenergic receptor antagonists propranolol and atenolol. Forskolin (10 microM) and the nonhydrolyzable cyclic AMP analogue 8-(4-chlorophenylthio)cyclic AMP (100 microM) had minimal effect on tritium efflux. However, phenylephrine-evoked increase in tritium efflux was dose dependently attenuated by the neuronal uptake blocker cocaine, and phenylephrine dose-dependently inhibited the incorporation of [3H]norepinephrine into neuronal stores. We conclude that the increase in tritium efflux induced by phenylephrine is independent of cyclic AMP accumulation and appears to be mediated by uptake of phenylephrine via the neuronal carrier-mediated amine transport process, which in turn promotes efflux of the adrenergic transmitter from its storage sites.

Effects of synthetic ANF (rANF(3-28)) on sympathetic neurotransmission in the isolated perfused rat kidney.

The effects of synthetic atrial natriuretic factor (rANF(3-28)) on sympathetic neurotransmission in the isolated perfused rat kidney was examined. ANF (10(-10)-10(-7) M) had no significant effect on stimulus-induced (1 Hz, 2 min) overflow of endogenous norepinephrine (NE) from the rat kidney. ANF also failed to affect stimulus-induced overflow which was markedly enhanced as a result of prejunctional beta-adrenoceptor activation with isoproterenol (10(-6)M). However, over the same concentration range ANF markedly attenuated the vasoconstrictor response to nerve stimulation. In addition, ANF significantly reduced the renal vasoconstrictor responses to intra-arterial injections of NE and angiotensin II. These results suggest that, while ANF potently inhibits renal sympathetic neurotransmission by inhibition of vascular responsiveness to vasoconstrictor stimuli, ANF does not appear to have a prejunctional effect to alter NE release from renal sympathetic nerves.

Renal periarterial nerve stimulation-induced vasoconstriction at low frequencies is primarily due to release of a purinergic transmitter in the rat.

In the isolated rat kidney, the vasoconstrictor response elicited by periarterial nerve stimulation at low frequencies (2 Hz) is resistant to alpha adrenergic receptor blockade. It has been proposed that in some blood vessels ATP is coreleased with norepinephrine during nerve stimulation to activate P2-purinergic receptors and is responsible for the component of the vasoconstrictor response that is resistant to alpha adrenergic receptor blockade. To assess the contribution of a purinergic transmitter in the vasoconstriction elicited by periarterial nerve stimulation in the isolated Tyrodes-perfused rat kidney, fractional overflow of [3H]norepinephrine and vasoconstrictor responses to renal nerve stimulation were examined after alpha adrenergic receptor blockade and/or P2-purinergic receptor desensitization. The alpha-1 adrenergic receptor antagonists prazosin (0.1-1.0 microM) and corynanthine (0.1-1.0 microM) and the nonselective alpha adrenergic receptor antagonist phentolamine (0.1-1.0 microM) did not significantly reduce vasoconstrictor responses elicited by low frequency (0.5-4 Hz) but attenuated the responses to high-frequency (6-10 Hz) periarterial nerve stimulation. At low-frequency renal nerve stimulation, selective P2-purinergic receptor desensitization abolished the vasoconstriction at 0.5 Hz and dramatically attenuated the responses up to 4 Hz. In the presence of prazosin, the component of the vasoconstrictor response that was resistant to alpha adrenergic receptor blockade at all frequencies of renal nerve stimulation was abolished after treatment with alpha, beta-methylene ATP. On the other hand, in the isolated perfused rabbit kidney, prazosin (1.0 microM) alone reduced dramatically the vasoconstrictor responses to periarterial nerve stimulation over the same frequencies used in the rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute epinephrine treatment fails to facilitate stimulus-induced neurotransmitter overflow in the isolated perfused rat kidney.

The effects of acute in vitro epinephrine treatment (40 nM, 1 hr) on stimulus-induced neurotransmitter overflow from the isolated perfused kidney were examined. This treatment resulted in the release of both norepinephrine and epinephrine upon nerve stimulation but no increase in fractional stimulus-induced overflow. Experiments utilizing beta and/or alpha adrenoceptor blockade revealed that beta adrenoreceptor blockade alone had no effect on overflow but that a dose-dependent decrease in stimulus-induced overflow was produced by beta adrenoceptor blockade after alpha adrenoceptor blockade. Thus, under normal conditions the facilitatory influence of epinephrine via prejunctional beta adrenoceptors is masked by the prejunctional alpha adrenoceptor negative feedback loop. The effects of extraneuronal presentation of isoproterenol, salbutamol and epinephrine on stimulus-induced neurotransmitter overflow also were examined. In the absence of alpha adrenoceptor blockade, only epinephrine failed to enhance neurotransmitter overflow. After competitive alpha adrenoceptor blockade, all three agents produced significant increases in stimulus-induced neurotransmitter overflow from the rat kidney, although alpha adrenoceptor stimulation appeared to limit this effect at high agonist concentrations. We conclude that the dominance of prejunctional alpha adrenoceptors in the rat kidney results in the inability of epinephrine to produce beta adrenoceptor-mediated facilitation of neurotransmitter overflow even when it is incorporated into renal neurotransmitter stores and coreleased with endogenous norepinephrine upon nerve stimulation.