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In cultured bovine adrenal chromaffin cells expressing Nav1.7 sodium channel isoform, we previously showed that veratridine-induced Na+ influx via Nav1.7 and the subsequent Ca2+ influx via voltage-dependent calcium channels activated protein kinase C-alpha and Akt, which converged on increasing inhibitory Ser9-phosphorylation of glycogen synthase kinase-3beta, decreasing constitutive Ser396-phosphorylation of tau. Here, veratridine increased constitutive Tyr204-phosphorylation of extracellular signal-regulated kinase-1/-2 (ERK1/ERK2) and constitutive Thr180/Tyr182-dual phosphorylation of p38 by approximately 118% (EC50=2.8 microM). Veratridine-induced increased phosphorylation levels of ERK1/ERK2 and p38 were abolished by tetrodotoxin, extracellular Ca2+ removal, or Gö6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole;Go6976] (protein kinase C-alpha inhibitor). PD98059 (2'-amino-3'-methoxyflavone) (ERK1/ERK2 inhibitor) or SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] (p38 inhibitor) attenuated veratridine-induced increased phosphorylation of glycogen synthase kinase-3beta and decreased phosphorylation of tau by approximately 54% and approximately 56%, as partial blockade by Gö6976. Additionally, basal constitutive phosphorylation levels of ERK1/ERK2 and p38 were decreased by PD98059 or SB203580, but not by SB216763 [3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indolo-3-yl)-1H-pyrrole-2,5-dione] (glycogen synthase kinase-3beta inhibitor) or extracellular Ca2+ removal. In this condition, PD98059 or SB203580 (but not SB216763 or extracellular Ca2+ removal) inhibited veratridine-induced 22Na+ influx and 45Ca2+ influx, without changing nicotine-induced 22Na+ influx via nicotinic receptor-associated cation channels and nicotine-induced 45Ca2+ influx via voltage-dependent calcium channels. These results suggest that Nav1.7-Ca2+ influx-protein kinase C-alpha pathway activated ERK1/ERK2 and p38, which increased phosphorylation of glycogen synthase kinase-3beta, decreasing tau phosphorylation. In veratridine-nontreated cells, basal constitutive activities of ERK1/ERK2 and p38 primed Nav1.7 to increase 22Na+ influx., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Evidence that Rho-kinase is involved in cerebral infarction has accumulated. However, it is uncertain whether Rho-kinase is activated in the brain parenchyma in cerebral infarction. To answer this question, we measured Rho-kinase activity in the brain in a rat cerebral infarction model. Sodium laurate was injected into the left internal carotid artery, inducing cerebral infarction in the ipsilateral hemisphere. At 6 h after injection, increase of activating transcription factor 3 (ATF3) and c-Fos was found in the ipsilateral hemisphere, suggesting that neuronal damage occurs. At 0.5, 3, and 6 h after injection of laurate, Rho-kinase activity in extracts of the cerebral hemispheres was measured by an ELISA method. Rho-kinase activity in extracts of the ipsilateral hemisphere was significantly increased compared with that in extracts of the contralateral hemisphere at 3 and 6 h but not 0.5 h after injection of laurate. Next, localization of Rho-kinase activity was evaluated by immunohistochemical analysis in sections of cortex and hippocampus including infarct area 6 h after injection of laurate. Staining for phosphorylation of myosin-binding subunit (phospho-MBS) and myosin light chain (phospho-MLC), substrates of Rho-kinase, was elevated in neuron and blood vessel, respectively, in ipsilateral cerebral sections, compared with those in contralateral cerebral sections. These findings indicate that Rho-kinase is activated in neuronal and vascular cells in a rat cerebral infarction model, and suggest that Rho-kinase could be an important target in the treatment of cerebral infarction.

In cultured bovine adrenal chromaffin cells, where Akt1 is the predominant isoform over Akt2 and Akt3, chronic (> or =12 h) treatment with 1-20 mM LiCl, an inhibitor of glycogen synthase kinase-3, decreased Akt1 level by approximately 52% (EC50=3.7 mM; t1/2=l2 h); it was associated with LiCl-induced increased levels of Ser9-phosphorylated glycogen synthase kinase-3beta (approximately 37%) and beta-catenin (approximately 59%), two hallmarks of glycogen synthase kinase-3beta inhibition. The same LiCl treatment did not change phosphoinositide 3-kinase, phosphoinositide-dependent kinase 1, and extracellular signal-regulated kinase-1/2 levels. Treatment with SB216763 [3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione], a selective inhibitor of glycogen synthase kinase-3, lowered Akt1 level by approximately 67% (EC50=2 microM; t1/2=l2 h), when SB216763 caused concentration- and time-dependent increase of beta-catenin level by approximately 76%. LiCl- or SB216763-induced Akt1 decrease, as well as increases of Ser9-phosphorylated glycogen synthase kinase-3beta and beta-catenin were restored to the control levels of nontreated cells after the washout of LiCl (20 mM for 24 h)- or SB216763 (30 microM for 24 h)-treated cells. LiCl-induced Akt1 reduction was not prevented by beta-lactone, lactacystin (two inhibitors of proteasome), calpastatin (an inhibitor of calpain), or leupeptin (an inhibitor of lysosome). LiCl decreased Akt1 mRNA level by 20% at 6 h, with no effect on Akt1 mRNA stability. These results suggest that glycogen synthase kinase-3beta inhibition caused down-regulation of Akt1 mRNA and Akt1 protein levels; conversely, constitutive activity of glycogen synthase kinase-3beta maintains steady-state level of Akt1 in quiescent adrenal chromaffin cells.

Using a continuous superfusion system of dog renal cortical slices, we studied the role of Ca(2+) in the intracellular control mechanism for renin release. The calcium ionophore A23187 (10 microM) produced a significant decrease in renin release. This effect was abolished in the absence of extracellular Ca(2+). Moreover, pretreatment with the calmodulin inhibitor W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, 20 microM) completely prevented the inhibitory effect of A23187 (10 microM). The beta-adrenoceptor agonist isoproterenol (1, 10 and 100 microM) produced a concentration-dependent increase in renin release. Pretreatment with W-7 (20 microM) potentiated the stimulation of renin release induced by isoproterenol (1 microM). These results suggest that A23187-induced inhibition of renin release is mediated by the activation of calmodulin via an increase in intracellular Ca(2+) and beta-adrenoceptor-stimulated renin release is modulated by intracellular Ca(2+) mobilization.

This study was designed to investigate possible effects of the Rho-kinase inhibitor, fasudil, on the progression of renal failure in rats with unilateral ureteral obstruction. The renal failure markers monitored were the extent of renal interstitial fibrosis and that of macrophage infiltration. In kidneys with unilateral ureteral obstruction, interstitial fibrosis was observed, using Sirius-Red staining, on day 16 after unilateral ureteral obstruction. Macrophage infiltration was observed by immunohistochemistry, using the antibody, ED1. Interstitial fibrosis and macrophage infiltration were significantly attenuated in fasudil-treated animals. The migration of monocytes in vitro elicited by N-formyl-methionyl-leucyl-phenylalanine was potently inhibited by fasudil and its active metabolite, hydroxyfasudil. These results suggest that inhibition of Rho-kinase produces a reduction of macrophage infiltration and represents a new therapeutic strategy for renal fibrosis, a major factor in the progression to end-stage renal failure., (Copyright 2002 Elsevier Science B.V.)

The role of Zn(2+) in oxidative stress during endotoxemia was investigated. In rats fed a Zn(2+)-deficient diet (Zn(2+) concentration of less than 1.5 mg/kg) for 8 weeks, the Zn(2+) level in the serum was about 62% lower than that in rats fed a Zn(2+)-adequate diet (Zn(2+) concentration, 50 mg/kg). The Zn(2+) level in serum 18 h after administration of endotoxin (6 mg/kg, i.p.) to Zn(2+)-deficient diet rats was markedly lower than that of the endotoxin/Zn(2+)-adequate diet group. Lipid peroxide formation in the liver of Zn(2+)-deficient diet rats was markedly increased 18 h after endotoxin injection compared with that in the endotoxin/Zn(2+)-adequate diet group. Metallothionein in the liver of endotoxin/Zn(2+)-adequate diet rats was increased more than 17-fold by endotoxin administration, while a markedly lower level of metallothionein was observed in the endotoxin/Zn(2+)-deficient diet group. On the other hand, treatment with ZnSO(4) (100 microM) significantly increased endotoxin (1 microg/ml)-induced tumor necrosis factor-alpha (TNF-alpha) production in J774A.1 cells. Our results clearly demonstrated that treatment with ZnSO(4) significantly inhibited the endotoxin-induced increase in intracellular Ca(2+) level in J774A.1 cells. However, a cell membrane-permeable Zn(2+) chelator, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN, 1 microM), did not affect the endotoxin-induced TNF-alpha production or Ca(2+) level in J774A.1 cells. In addition, we investigated whether Zn(2+) can suppress nitric oxide (NO) generation and cytotoxicity in endotoxin-treated cells. Treatment with ZnSO(4) (50 microM) significantly inhibited endotoxin-induced NO production in J774A.1 cells, but did not affect endotoxin-induced cytotoxicity. These findings suggest that zinc may play an important role, at least in part, in the oxidative stress during endotoxemia., (Copyright 2002 Elsevier Science B.V.)

We eluciated whether K+ channels modulate adrenal catecholamine secretion induced by pituitary adenylate cyclase-activating polypeptide (PACAP) in the isolated perfused rat adrenal gland. PACAP (100 nM) increased adrenal epinephrine output. The PACAP-induced responses were enhanced by treatment with apamin (10-100 nM) in a concentration-dependent manner. In the presence of nifedipine (3 microM), apamin (1 microM) did not enhance the PACAP-induced responses. Charybdotoxin (1-100 nM) had little influence on the PACAP-induced responses. These results suggest that small-conductance Ca2+-activated K+ channels interfere with L-type voltage-dependent Ca2+ channels to counteract the PACAP-induced adrenal catecholamine secretion.

The effects of 6,7,8, 9-tetrahydro-2-methyl-5H-cyclohepta[b]pyridine-3-carbonylguanidine maleate (TY-12533) on myocardial ischemia/reperfusion injury were evaluated in rats. Inhibitory effects of TY-12533, TY-50893 (the 9-chloro derivative of TY-12533) and cariporide on the platelet Na(+)/H(+) exchanger in vitro were almost equal at pH 6.2 and decreased at pH 6.7; but TY-12533 was four times more potent than TY-50893 and cariporide at pH 6.7. TY-12533, TY-50893 and cariporide administered before ischemia (0.01-1 mg/kg, i.v.) suppressed the ischemia/reperfusion-induced arrhythmias to the same extent in vivo; but TY-12533 was more effective than cariporide and TY-50893 when they were administered during ischemia (0.1-1 mg/kg). Similar results were obtained for the inhibitory effects of these drugs administered before ischemia (0.03-0.1 mg/kg, i.v.) and during ischemia (0.1-1 mg/kg) on the ischemia/reperfusion-induced myocardial infarction. These differences between TY-12533 and the other drugs in vitro and in vivo may be ascribed to the pK(a) values of the guanidinium moiety of TY-12533 (6.93), TY-50893 (6.35) and cariporide (6.28).

Intrarenal arterial infusion of endothelin-1 (1, 3 and 10 ng/kg per min) reduced renal blood flow, urine flow rate and urinary Na+ excretion without affecting fractional Na+ excretion in anesthetized rabbits. An endothelin ET(A) receptor antagonist (R)2-[(R)-2-[(S)-2-[[1-(hexahydro-1H-azepinyl)]carbonyl]amino-4-me thyl-pentanoyl]amino-3-[3-(1-methyl-1H-indolyl)]propionyl]amino-3-(2-pyr idyl)propionic acid (FR139317, 1 microg/kg per min) attenuated the endothelin-1 (1 ng/kg per min)-induced renal responses. An endothelin ET(B) receptor antagonist N-cis 2,6-dimetylpiperidinocarbonyl-L-gamma-metylleucyl-D-1-met hoxycarbonyltryptophanyl-D-norleucine (BQ-788, 1 microg/kg per min) potentiated the endothelin-1-induced changes in renal blood flow, urine flow rate and urinary Na+ excretion. A nitric oxide (NO) synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME, 50 microg/kg per min) also potentiated the endothelin-1-induced reductions in urine flow rate and urinary Na+ excretion but not the reduction in renal blood flow. Endothelin-1 reduced fractional Na+ excretion in the presence of BQ-788 or L-NAME. A spontaneous NO donor 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene (30 ng/kg per min) slightly attenuated the antinatriuresis but not the vasoconstriction induced by endothelin-1. These results suggest that in the rabbit kidney in vivo endothelin ET(A) receptors mediate endothelin-1-evoked vasoconstriction and tubular Na+ reabsorption, that the concomitant stimulation of endothelin ET(B) receptors by endothelin-1 counteracts both the ET(A) receptor-mediated vascular and tubular actions, and that the tubular action, but not the vascular action, of endothelin-1 is also susceptible to changes in renal NO level.

Intrarenal arterial infusion of C-type natriuretic peptide (CNP, 50 ng/kg per min) increased urine flow rate without affecting glomerular filtration rate. Intrarenal arterial bolus injection of angiotensin II (25, 50 and 100 ng) or of norepinephrine (0.25, 0.5 and 1.0 microg) reduced renal blood flow. The blood flow response induced by angiotensin II was slightly attenuated but the response induced by norepinephrine was unaffected during CNP infusion. These results suggest that exogenous CNP, even at the pharmacological dose that can induce diuresis, has little effect on the canine renal vasculature.

Intrarenal arterial infusion of angiotensin II (4 ng/kg per min) reduced glomerular filtration rate and urinary Na+ excretion without affecting fractional Na+ excretion. Infusion of norepinephrine (30 ng/kg per min) reduced both urinary Na+ excretion and fractional Na+ excretion with a slight hypofiltration. The angiotensin II- and the norepinephrine-induced renal responses were suppressed during simultaneous infusion of a spontaneous nitric oxide donor 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl 1-triazene (NOC 7, 30 ng/kg per min) which itself had little influence on the renal parameters. The results suggest that in the rabbit kidney in vivo NOC 7 can interfere with the angiotensin II-induced hypofiltration and norepinephrine-evoked tubular reabsorption and thereby suppresses their antinatriuretic actions.

Intrarenal arterial infusion of angiotensin II (4 ng/kg/min) reduced renal blood flow, glomerular filtration rate and urinary Na+ excretion (UNaV) without affecting fractional Na+ excretion (FENa) in anesthetized rabbits. Losartan (10 micrograms/kg/min) abolished these angiotensin II-induced renal responses. The renal blood flow, glomerular filtration rate and UNaV responses were potentiated during intrarenal arterial infusion of N omega-nitro-L-arginine methyl ester (L-NAME, 10 micrograms/kg/min). A high dose of L-NAME (50 micrograms/kg/min) also potentiated the renal blood flow and UNaV responses but not the glomerular filtration rate response. Angiotensin II reduced FENa during L-NAME infusion at either dose. In L-NAME-pretreated rabbits, losartan abolished the angiotensin II-induced renal blood flow and glomerular filtration rate responses, but the reduction in FENa still remained. The present study suggests that in the rabbit kidney (1) nitric oxide attenuates the angiotensin II-induced (angiotensin AT1 receptor-mediated) vasoconstriction and (2) angiotensin II can evoke losartan-resistant tubular Na+ reabsorption, but the tubular action is concealed by nitric oxide.

Atrial natriuretic peptide (ANP, 10 and 50 ng/kg per min), infused into the renal artery, suppressed decreases in renal blood flow induced by intrarenal arterial injection of angiotensin II (Ang II, 25-100 ng) and norepinephrine (NE, 0.25-1 microgram) in anesthetized dogs. Sodium nitroprusside (SNP, 0.1-5.0 micrograms/kg per min) slightly attenuated the blood flow response to Ang II but not the response to NE. 8-Bromo cyclic GMP (8bcGMP, 0.5-25 micrograms/kg per min) did not suppress the blood flow response to Ang II. Although at a high dose ANP attenuated the blood flow response to Bay K 8644 (1-4 micrograms), nifedipine pretreatment (20 micrograms/kg plus 1 microgram/kg per min i.v.) did not affect the inhibitory effect of ANP on the NE-induced response. The vaso-inhibitory effects of ANP therefore could not be related exclusively to stimulation of cGMP production or inhibition of voltage-dependent Ca2+ channels.

We examined the possible prophylactic potential of HA1077, a calcium antagonist and vasodilator, on chronic cerebral vasospasm induced in a two-hemorrhage canine model, and also its effects on cerebral hemodynamics. The intravenous infusion of HA1077 3 mg/kg over 30 min twice daily (day 1-day 7) after the first intracisternal injection of 5 ml autologous blood significantly prevented the occurrence of chronic cerebral vasospasm. The mean diameter of the basilar arteries on day 7 was 66.1 +/- 1.6% (n = 7) of the baseline before the intracisternal injection of blood, compared to 54.2 +/- 1.6% (n = 9) of the baseline in the untreated group (P less than 0.01). Bolus intravenous administration of HA1077 (0.1 and 0.3 mg/kg) dose dependently increased local cerebral blood flow. Since HA1077 prevents the development of chronic cerebral vasospasm after subarachnoid hemorrhage and improves hemodynamic functions, as manifested by increases in local cerebral blood flow, further study is warranted regarding the possible clinical use of this drug.

To seek evidence for the involvement of acetylcholinesterase activity in the modulatory influence of the airway epithelium, we examined responses to acetylcholine (ACh), bethanechol, histamine or KCl in isolated epithelium-intact and epithelium-denuded guinea-pig trachealis preparations. The concentration-response curves to ACh were shifted 26-fold to the left by epithelial denudation but the contractile response to KCl was not altered. The response to histamine in epithelium-denuded preparations increased 4-fold with no attenuation in the presence of physostigmine (30 nM). Physostigmine (30 nM) potentiated the response to ACh in epithelium-intact tissues more (about 26-fold) than in epithelium-denuded tissues (about 3.5-fold). Thus, in the presence of physostigmine removing the epithelium had only a slight effect (not statistically significant) on the potency of ACh to contract the trachea. Removing the epithelium had no effect on the potency of bethanechol, a muscarinic receptor agonist that is not a substrate for cholinesterases. Physostigmine itself contracted the trachealis muscle but the pD2 values and maximum responses in epithelium-intact and denuded preparations were not significantly different. The frequency-response curves to electrical field-stimulated cholinergic contractions were unaffected by removing the epithelium. In conclusion, the principal mechanism by which the epithelium inhibits contraction of guinea-pig trachea to exogenously applied ACh is via epithelium-derived acetylcholinesterase activity.

Endothelin infused into the renal artery (0.3-3 ng/kg per min) of anesthetized dogs suppressed the increases in renal venous plasma norepinephrine (NE) concentration and calculated NE efflux induced by renal nerve stimulation at low frequencies (0.7-1.2 Hz) but not at high frequencies (2-3.5 Hz). Endothelin failed to affect the decrease in renal blood flow induced by renal nerve stimulation or exogenous NE. These findings suggest that endothelin inhibits NE release during a slight rise in renal nerve activity, while it does not affect alpha-adrenoceptor-mediated vasoconstriction in the dog kidney.

The effects of atrial natriuretic peptide (ANP) on the neural control of renal blood flow were examined in anesthetized dogs. Intrarenal arterial infusion of ANP (alpha-hANP, 10 and 50 ng/kg per min) suppressed the decrease in renal blood flow but not the increase in renal venous plasma norepinephrine concentration induced by renal nerve stimulation (1 and 2 Hz, for 1 min). ANP also attenuated the blood flow response to intrarenal arterial injection of methoxamine (5-20 micrograms). These results suggest that ANP acts at a postsynaptic site to suppress adrenergically induced vasoconstriction in the dog kidney.

The effect of serotonin (5-HT) on renin release was examined in denervated kidney of the pentobarbital-anesthetized dog. The intrarenal arterial infusion of a large dose of 5-HT (1 micrograms/kg per min) increased the renin secretion rate with an initial decrease and a subsequent increase in renal blood flow. Systemic blood pressure or heart rate was unaffected. The renin release induced by 5-HT was suppressed during intrarenal arterial infusion of a 5-HT1 and 5-HT2 antagonist, methysergide (30 micrograms/kg per min), or a selective 5-HT2 antagonist, ketanserin (3 micrograms/kg per min). A cyclooxygenase inhibitor, indomethacin (5 mg/kg i.v.), also suppressed the 5-HT-induced renin release. These results suggest that stimulation of renal 5-HT receptors, probably of the 5-HT2 type, can induce renin release from the dog kidney, which may be dependent on renal prostaglandin production. The present results, however, do not allow us to conclude that the renal 5-HT receptors play a physiological role in the control of renin release.

1-(5-Isoquinolinesulfonyl)-homopiperazine, HA1077, is a calcium antagonist with anti-vasospastic properties. This compound blocks intracellular actions of calcium in a variety of experiments. In the present study, we examined the effects of HA1077 on the vascular actions of endothelin, an endothelium-derived vasoactive peptide, in dogs in vitro and in vivo. Intracisternal injections of endothelin (0.01 nmol) produced a significant vasospasm, as measured by angiography, similar to that seen in the canine hemorrhage model. Infusion of HA1077 led to a significant dilatation of the spastic basilar artery in endothelin-treated dogs. The rank order of in vitro contractile activity in canine cerebral arteries was a stable thromboxane A2 analog greater than endothelin greater than 5-hydroxytryptamine greater than prostaglandin F2 alpha greater than histamine greater than noradrenaline. HA1077 effectively antagonized the endothelin-induced contraction of canine basilar arterial strips in both calcium-containing and calcium-free medium. The present results indicate that HA1077 is an effective antagonist for endothelin in vitro and in vivo.

The alpha 1-adrenoceptor antagonistic effect of SGB-1534, a novel phenylpiperazine derivative, was examined in the renal vascular bed of pentobarbital-anesthetized dogs. Renal nerve stimulation (RNS, 1 ms duration, 2, 3 and 4 Hz) or intrarenal bolus injection of methoxamine (0.5, 1 and 2 micrograms/kg) or guanabenz (1, 3 and 10 micrograms/kg) produced a frequency- or dose-dependent decrease in renal blood flow (RBF). Both the RBF responses to RNS and methoxamine were inhibited dose dependently by an intrarenal infusion of SGB-1534 (1-30 ng/kg per min) or prazosin (30-300 ng/kg per min). When the equipotent inhibitory doses of the antagonists were compared, the antagonistic potency of SGB-1534 on the RBF responses evoked by RNS and methoxamine was about 30 times greater than that of prazosin. Prazosin also attenuated the RBF response to guanabenz, whereas SGB-1534 had little effect. These results suggest that SGB-1534 has a selective alpha 1-adrenoceptor-blocking property and that it inhibits neurally mediated renal vasoconstriction. The alpha 1-adrenoceptor antagonistic potency and selectivity of SGB-1534 may be greater than that of prazosin.

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.

The effects of prazosin and yohimbine on sympathetic neurotransmission to the heart were investigated in perfused dog hearts in situ in an attempt to determine whether alpha 1-adrenoceptors are located presynaptically in the cardiac sympathetic nerves. Intra-arterial injections of prazosin (1-30 micrograms) and yohimbine (0.3-10 micrograms) into the right coronary artery during cardiac sympathetic nerve stimulation further increased the tachycardia resulting from the stimulation. Continuous infusions of methoxamine (20-40 micrograms/min) and of clonidine (2-4 micrograms/min) into the right coronary artery during cardiac sympathetic nerve stimulation caused sustained reduction of the tachycardia. Prazosin under methoxamine infusion enhanced the tachycardia to a greater extent than in the absence of methoxamine. Prazosin under clonidine infusion enhanced the tachycardia to the same extent as it did in the absence of clonidine. These results suggest that prazosin antagonizes the effect of methoxamine but does not antagonize that of clonidine. The results obtained with yohimbine were in contrast to the effects of prazosin, showing the antagonism of clonidine by yohimbine. Prazosin and yohimbine both had little effect on the heart rate during either the resting state or the infusion of norepinephrine. These results suggest that the prazosin- and yohimbine-induced enhancement of the tachycardia resulting from cardiac sympathetic nerve stimulation is due to a presynaptic effect. However, the presynaptic effect of prazosin appears to differ from that of yohimbine. The presence of presynaptic alpha 1-adrenoceptors regulating norepinephrine release, as well as of alpha 2-adrenoceptors, is suggested in the cardiac sympathetic nerves of the dog.

Chemical removal of the endothelium by saponin in the isolated dog femoral artery was investigated by comparing the relaxant responses to endothelium-dependent and -independent vasodilators of saponin-treated rings with the responses of non-treated rings. Saponin treatment was done by incubating rings with Krebs-Henseleit solution containing 0.1, 0.3 or 1 mg/ml of saponin for 45 min at 37 degrees C. In non-treated rings, acetylcholine (10(-8)-3 X 10(-6) M) caused a concentration-dependent relaxation of rings precontracted with prostaglandin F2 alpha (3 X 10(-6) M). The acetylcholine-induced relaxation was reduced in rings pretreated with 0.1 mg/ml of saponin and almost abolished with 0.3 or 1 mg/ml. Prostaglandin F2 alpha-induced contraction was suppressed weakly by treatment with 0.3 mg/ml and markedly with 1 mg/ml saponin. The treatment with 0.3 mg/ml saponin markedly reduced relaxations caused by substance P (10(-9)-3 X 10(-8) M) and by Ca2+-ionophore A23187 (10(-6) M). Relaxant responses of saponin-treated rings to nitroglycerin and to nitroprusside were almost identical with those of non-treated rings. These results showing selective suppression by saponin of the endothelium-dependent relaxation suggest that saponin removes the endothelial cells from the intimal surface of the artery, and this was confirmed by electron microscopy. The endothelium removing method with saponin seems to be useful as a pharmacological tool for vascular investigations.

Effects of antidepressants and other drugs on the behaviour of rats in the forced swimming test were examined. Acute and chronic administration of antidepressants reduced the duration of immobility during the first 5 or 10 min of a 30 min test by prolonging escape-directed behaviour which appeared only during this period. However, the drugs did not affect the duration of immobility during the last 20 or 25 min, when the rats were in a state of almost complete immobility. In contrast, methamphetamine, caffeine and scopolamine reduced the duration of immobility not only during the first 5 min but also the next 15 or 25 min without prolonging the escape-directed behaviour but by increasing general motor activity. The effect of antidepressants was potentiated by chronic treatment as compared to acute administration. In the chronic experiments, a significant reduction in the duration of immobility was first observed on the 6th day of the treatment. Although a single injection of diphenhydramine caused an effect similar to antidepressants, this effect disappeared after chronic treatment. These results indicate that immobility itself is not affected by antidepressants. However, it is suggested that the reduction in the duration of immobility only during the first 5 min of the test, which was caused by the prolongation of the escape-directed behaviour, and the potentiation of the effect after chronic treatment are an action specific to antidepressants.

The effects of neuropeptide Y (NPY) on the vascular tone of isolated cerebral arteries and vertebral blood flow (VBF) were studied in dogs. NPY elicited a dose-dependent contraction of arteries derived from the brain with ED50 values of 2 nM for the middle cerebral and basilar arteries. Arteries from the neck did not respond to NPY. Intra-arterial administration of NPY as a bolus reduced the VBF dose dependently, with no significant alteration of mean arterial blood pressure and heart rate. The decrease in VBF developed slowly and had a long duration, which was consistent with the observations made in vitro. NPY suppressed the contractile effect of noradrenaline (NA) on isolated cerebral arteries and pretreatment with NPY suppressed the effect of NA on VBF, indicating that NPY contributes to the inhibitory modulation of postsynaptic adrenergic mechanisms. These findings suggest that NPY could have a role in the regulation of cerebral circulation.