Role of endogenous PACAP in catecholamine secretion from the rat adrenal gland.

We elucidated the contribution of endogenous pituitary adenylate cyclase-activating polypeptide (PACAP) to neurally evoked catecholamine secretion from the isolated perfused rat adrenal gland. Infusion of PACAP (100 nM) increased adrenal epinephrine and norepinephrine output. The PACAP-induced catecholamine output responses were inhibited by the PACAP type I receptor antagonist PACAP- (6-38) (30-3,000 nM) but were resistant to the PACAP type II receptor antagonist [Lys1,Pro2,5,Ara3,4,Tyr6]-vasoactive intestinal peptide (LPAT-VIP; 30-3,000 nM). Transmural electrical stimulation (ES; 1-10 Hz) or infusion of ACh (6-200 nM) increased adrenal epinephrine and norepinephrine output. PACAP-(6-38) (3,000 nM), but not LPAT-VIP, also inhibited the ES-induced catecholamine output responses. However, PACAP-(6-38) did not affect the ACh-induced catecholamine output responses. PACAP at low concentrations (0.3-3 nM), which had no influence on catecholamine output, enhanced the ACh-induced catecholamine output responses, but not the ES-induced catecholamine output responses. These results suggest that PACAP is released from the nerve endings to facilitate the neurally evoked catecholamine secretion through PACAP type I receptors in the rat adrenal gland.

Role of calcium channels and adenylate cyclase in the PACAP-induced adrenal catecholamine secretion.

We elucidated the functional contribution of voltage-dependent calcium channels (VDCCs) and adenylate cyclase to epinephrine (Epi) and norepinephrine (NE) secretion induced by pituitary adenylate cyclase-activating polypeptide (PACAP) in the isolated perfused rat adrenal gland. PACAP increased Epi and NE output, which was inhibited by perfusion with calcium-free solution or by nifedipine, an L-type VDCC blocker. However, the PACAP-induced responses were resistant to omega-conotoxin GVIA, an N-type VDCC blocker, or omega-conotoxin MVIIC, a P/Q-type VDCC blocker. MDL-12330A, an adenylate cyclase inhibitor, inhibited the PACAP-induced increase in Epi, but not NE, output. Treatment with nifedipine and MDL-12330A caused additive inhibition of the PACAP-induced catecholamine responses. These results suggest that opening of L-type VDCCs is responsible for adrenal catecholamine secretion induced by PACAP and that activation of adenylate cyclase is involved in the PACAP-induced Epi, but not NE, secretion. These pathways may act independently of each other.

Role of potassium channels in catecholamine secretion in the rat adrenal gland.

We elucidated the functional contribution of K(+) channels to cholinergic control of catecholamine secretion in the perfused rat adrenal gland. The small-conductance Ca(2+)-activated K(+) (SK(Ca))-channel blocker apamin (10-100 nM) enhanced the transmural electrical stimulation (ES; 1-10 Hz)- and 1, 1-dimethyl-4-phenyl-piperazinium (DMPP; 5-40 microM)-induced increases in norepinephrine (NE) output, whereas it did not affect the epinephrine (Epi) responses. Apamin enhanced the catecholamine responses induced by acetylcholine (6-200 microM) and methacholine (10-300 microM). The putative large-conductance Ca(2+)-activated K(+) channel blocker charybdotoxin (10-100 nM) enhanced the catecholamine responses induced by ES, but not the responses induced by cholinergic agonists. Neither the K(A) channel blocker mast cell degranulating peptide (100-1000 nM) nor the K(V) channel blocker margatoxin (10-100 nM) affected the catecholamine responses. These results suggest that SK(Ca) channels play an inhibitory role in adrenal catecholamine secretion mediated by muscarinic receptors and also in the nicotinic receptor-mediated secretion of NE, but not of Epi. Charybdotoxin-sensitive Ca(2+)-activated K(+) channels may control the secretion at the presynaptic site.

[Experimental pharmacology of the in vivo kidney: renal actions of cAMP-related drugs].

The kidney plays a pivotal role in cardiovascular homeostasis through control of extracellular fluid volume, the mechanisms of which have been studied in detail at both cellular and molecular levels in each portion of the kidney. Renal clearance experiments in vivo are useful for analyzing renal functions as the integrated system of vascular, glomerular and tubular components. Physiological responses and drug effects within the kidney can be more clearly analyzed by measuring renal release of endogenous substances and by applying intrarenal arterial drug administration. For example, our recent studies in anesthetized dogs revealed the participation of phosphodiesterase IV in regulation of renal cAMP level and the influences by changes in renal cAMP level on renal hemodynamics, glomerular filtration and tubular reabsorption. The in vivo experiments also allow us to evaluate a sequence of events in the neural control of renal functions. We observed that an adenylate cyclase activator counteracted hypofiltration to attenuate antinatriuresis during sympathetic activation without inhibiting neurotransmitter release. The experiments in the in vivo kidney, although they provide only indirect information on intracellular events, are necessary for understanding the total renal functions and could thereby contribute to physiology and pharmacology of the cardiovascular system.

[Control mechanisms of renal functions: effects of cardiovascular drugs on vasoconstrictive and antinatriuretic stimuli in the in vivo kidney].

The kidney contributes to cardiovascular homeostasis through Na+ and water excretion and renin secretion. Changes in renal functions, therefore, have a close relationship to pathophysiology of cardiovascular diseases and to drug efficacy for them. The functions of the kidney are controlled by the sympathetic nervous system and various kinds of humoral factors and by their complicated interactions. Studies in the intact and working kidney in vivo have been providing physiologically significant information on the renal functions and drug actions. This review, by demonstrating data obtained in our laboratory with experiments in anesthetized dogs in vivo, refers mainly to the neural control of renal functions and renal actions of atrial natriuretic peptide (ANP) and an adenylate cyclase activator NKH477, either of which could be used for the treatment of congestive heart failure. Electrical stimulation of the renal nerves, which could mimic the events during elevation of renal sympathetic nerve activity, induces frequency-dependent renal norepinephrine release, renal vasoconstriction, antinatriuresis and renin secretion. ANP causes potent natriuresis and suppresses the nerve stimulation-induced renin secretion and renal vasoconstriction without affecting the norepinephrine release. Effects of ANP on other vasoconstrictive and antinatriuretic stimuli such as angiotensin II and endothelin are also demonstrated. Renal actions of NKH477 had been unknown, but we revealed that NKH477 elevates renal cAMP level and causes vasodilation and natriuresis. NKH477 also suppresses the nerve stimulation-induced renal vasoconstriction, and thereby blunts the antinatriuresis. The renal actions of these drugs clarified in our study may contribute to their curative effects on congestive heart failure.

Effects of NKH477 on renal functions and cyclic AMP production in anesthetized dogs.

The present study was undertaken to evaluate the effects of an adenylate cyclase activator N,N-dimethyl-beta-alanine[3R-(3alpha, 4alphabeta, 5beta, 6beta, 6aalpha, 10alpha, 10abeta, 10balpha)]-5(acetyloxy)-3-ethenyldodecahydro-10, 10b-dihydroxy-3, 4a, 7, 7, 10a-pentamethyl-1-oxo-1H-naphtho [2,1-b] pyran-6-yl ester hydrochloride (NKH477) on renal functions and cyclic AMP production in the dog kidney. The intrarenal arterial infusion of NKH477 (30, 100 and 300 ng kg(-1) min(-1)) increased renal blood flow, glomerular filtration rate, urine flow rate, urinary Na+ and cyclic AMP excretion, fractional Na+ excretion and arterial and renal venous plasma cyclic AMP concentrations in a dose-dependent manner. The intrarenal arterial infusion of rolipram (0.3 microg kg(-1) min(-1)), a cyclic AMP-specific phosphodiesterase inhibitor, also caused the same renal responses as NKH477. The increasing effects of NKH477 on renal blood flow, fractional Na+ excretion and renal venous plasma cyclic AMP concentration were facilitated in the presence of rolipram. NKH477 reduced glomerular filtration rate and filtration fraction in the presence of rolipram. The increasing effects of NKH477 on urine flow rate and urinary Na+ excretion were not affected by rolipram. The present results suggest that NKH477 increases glomerular filtration and suppresses tubular sodium reabsorption through activation of cyclic AMP production, and thereby induces natriuresis. The results also demonstrate that renal cyclic AMP level during the activation of adenylate cyclase is regulated by phosphodiesterase IV in both the vascular and tubular sites.

Effects of rolipram and cilostamide on renal functions and cyclic AMP release in anesthetized dogs.

The present study was undertaken to examine whether phosphodiesterases III and IV regulate renal cAMP level and whether inhibition of these enzymes influences renal functions in anesthetized dogs. The intrarenal arterial infusion of rolipram (0.1, 0.3, and 1 microgram/kg/min), a selective phosphodiesterase IV inhibitor, increased renal blood flow, glomerular filtration rate, urine flow rate, and urinary Na+ excretion with elevating arterial and renal venous plasma cAMP concentrations and urinary cAMP excretion. However, cilostamide (0.1, 0.3, and 1 microgram/kg/min), a selective phosphodiesterase III inhibitor, did not affect the values of these parameters. Indomethacin (3 mg/kg i.v. bolus and 1 mg/kg/min i.v. infusion), a cyclooxygenase inhibitor, reduced the basal arterial and renal venous plasma cAMP concentrations and blunted the rolipram-induced elevation of cAMP concentrations and urinary cAMP excretion. The effects of rolipram on renal hemodynamics and urine formation were attenuated in the presence of indomethacin. These results suggest that in the dog kidney in vivo, 1) phosphodiesterase IV, but not phosphodiesterase III, participates in degradation of cAMP and 2) the inhibition of phosphodiesterase IV enhances glomerular filtration and urinary Na+ excretion, the responses of which depend in part on indomethacin-susceptible (prostaglandin-mediated, probably) control of basal cAMP level.

Effects of adrenomedullin and PAMP on adrenal catecholamine release in dogs.

We examined the effects of proadrenomedullin-derived peptides on the release of adrenal catecholamines in response to cholinergic stimuli in pentobarbital sodium-anesthetized dogs. Drugs were administered into the adrenal gland through the phrenicoabdominal artery. Splanchnic nerve stimulation (1, 2, and 3 Hz) and ACh injection (0.75, 1.5, and 3 microgram) produced frequency- or dose-dependent increases in adrenal catecholamine output. These responses were unaffected by infusion of adrenomedullin (1, 3, and 10 ng. kg-1. min-1) or its selective antagonist adrenomedullin-(22-52) (5, 15, and 50 ng. kg-1. min-1). Proadrenomedullin NH2-terminal 20 peptide (PAMP; 5, 15, and 50 ng. kg-1. min-1) suppressed both the splanchnic nerve stimulation- and ACh-induced increases in catecholamine output in a dose-dependent manner. PAMP also suppressed the catecholamine release responses to the nicotinic agonist 1, 1-dimethyl-4-phenylpiperazinium (0.5, 1, and 2 microgram) and to muscarine (0.5, 1, and 2 microgram), although the muscarine-induced response was relatively resistant to PAMP. These results suggest that PAMP, but not adrenomedullin, can act as an inhibitory regulator of adrenal catecholamine release in vivo.

Effects of sodium nitroprusside on renal functions and NO-cGMP production in anesthetized dogs.

Although the renal nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) system plays an important role in maintaining urinary sodium and water excretion, effects of an authentic NO donor sodium nitroprusside (SNP) on urine formation have been controversial. In this study, we examined whether SNP increases renal NO release and cGMP production and induces natriuresis in the denervated kidney of anesthetized dogs. The intrarenal arterial infusion of SNP at 10, 30, and 100 ng/kg/min did not affect renal function or NO-cGMP production. The higher dose of SNP (1,000 ng/kg/min) reduced systemic blood pressure and urine flow rate. The antidiuresis was observed also in the contralateral control kidney, the degree of which was larger than that observed in the ipsilateral SNP-infused kidney. During the SNP infusion, reductions in urinary Na+ excretion, fractional Na+ excretion, and urinary nitrite + nitrate excretion occurred in the control kidney but not in the SNP-infused kidney. Urinary cGMP excretion and renal venous plasma cGMP concentration were significantly increased during the SNP infusion in the SNP-infused kidney but not in the control kidney. These renal effects of SNP were similar to those obtained by intrarenal arterial infusion of a specific NO donor, NOC 7 (300 ng/kg/min). These results suggest that SNP can produce nitric oxide and increase cGMP levels in the kidney and suppress sodium reabsorption, but the natriuretic property of SNP may be masked by its counteracting effects including the systemic hypotension in anesthetized dogs.

Role of K+ channels in adrenal catecholamine secretion in anesthetized dogs.

We examined the role of K+ channels in the secretion of adrenal catecholamine (CA) in response to splanchnic nerve stimulation (SNS), acetylcholine (ACh), 1,1-dimethyl-4-phenyl-piperazinium (DMPP), and muscarine in anesthetized dogs. K+ channel blockers and the cholinergic agonists were infused and injected, respectively, into the adrenal gland. The voltage-dependent K+ channel (KA type) blocker mast cell degranulating (MCD) peptide infusion (10-100 ng/min) enhanced increases in CA output induced by SNS (1-3 Hz), but it did not affect increases in CA output induced by ACh (0.75-3 micrograms), DMPP (0.1-0.4 microgram), or muscarine (0.5-2 micrograms). The small-conductance Ca(2+)-activated K+ (SKCa) channel blocker scyllatoxin infusion (10-100 ng/min) enhanced the ACh-, DMPP-, and muscarine-induced increases in CA output, but it did not affect the SNS-induced increases in CA output. These results suggest that KA channels may play an inhibitory role in the regulation of adrenal CA secretion in response to SNS and that SKCa channels may play the same role in the secretion in response to exogenously applied cholinergic agonists.

Effects of adenosine on adrenergically induced renal vasoconstriction in dogs.

The role of exogenous and endogenous adenosine in the neural control of renal blood flow was studied in anesthetized dogs. The plasma norepinephrine (NE) concentration was measured by high-performance liquid chromatography and the renal NE secretion rate was calculated. Renal nerve stimulation (1-3 Hz) reduced renal blood flow and increased NE secretion rate. The intrarenal arterial injection of NE (0.3-1.0 micrograms) also reduced renal blood flow. Infusion of adenosine (10-100 micrograms/min) into the renal artery attenuated the increase in NE secretion rate induced by renal nerve stimulation, but the nerve stimulation-induced decrease in renal blood flow was unaffected. On the other hand, adenosine potentiated the NE-induced renal blood flow response. Similar results were obtained with an adenosine potentiator, dipyridamole (1-10 micrograms/min). An adenosine receptor blocker, theophylline (0.3-1.0 mg/min), potentiated the NE secretion rate response induced by nerve stimulation, without any change in the renal blood flow response. The NE-induced renal blood flow response was attenuated by theophylline. These results suggest that adenosine inhibits neural NE release and enhances vasoconstriction in the dog kidney during sympathetic stimulation under in vivo conditions. These post- and presynaptic mechanisms may thus be activated by endogenous adenosine.