Calcium recruitment in renal vasculature: NE effects on blood flow and cytosolic calcium concentration.
(25/2286)
This study provides new information about the relative importance of Ca2+ mobilization and entry in the renal vascular response to adrenoceptor activation. We measured renal blood flow (RBF) in Sprague-Dawley rats in vivo using electromagnetic flowmetry. We measured intracellular free Ca2+ concentration ([Ca2+]i) in isolated afferent arterioles utilizing ratiometric photometry of fura-2 fluorescence. Renal arterial injection of NE produced a transient decrease in RBF. The response was attenuated, in a dose-dependent manner, up to approximately 50% by nifedipine, an antagonist of L-type Ca2+ entry channels. Inhibition of Ca2+ mobilization by 3,4, 5-trimethoxybenzoic acid-8-(diethylamino)octyl ester (TMB-8) inhibited the renal vascular effects of NE in a dose-dependent manner, with maximal blockade of approximately 80%. No additional attenuation was observed when nifedipine and TMB-8 were administered together. In microdissected afferent arterioles, norepinephrine (NE; 10(-6) M) elicited an immediate square-shaped increase in [Ca2+]i, from 110 to 240 nM. This in vitro response was blocked by nifedipine (10(-6) M) and TMB-8 (10(-5) M) to a degree similar to that of the in vivo experiments. A nominally calcium-free solution blocked 80-90% of the [Ca2+]i response to NE. The increased [Ca2+]i elicited by depolarization with medium containing 50 mM KCl was totally blocked by nifedipine. In contrast, TMB-8 had no effect. Our results indicate that both Ca2+ entry and mobilization play important roles in the renal vascular Ca2+ and contractile response to adrenoceptor activation. The entry and mobilization mechanisms activated by NE may interact. That a calcium-free solution caused a larger inhibition of the NE effects on afferent arterioles than nifedipine suggests more than one Ca2+ entry pathway. (+info)
Calcium antagonists ameliorate ischemia-induced endothelial cell permeability by inhibiting protein kinase C.
(26/2286)
BACKGROUND: Dihydropyridines block calcium channels; however, they also influence endothelial cells, which do not express calcium channels. We tested the hypothesis that nifedipine can prevent ischemia-induced endothelial permeability increases by inhibiting protein kinase C (PKC) in cultured porcine endothelial cells. METHODS AND RESULTS: Ischemia was induced by potassium cyanide/deoxyglucose, and permeability was measured by albumin flux. Ion channels were characterized by patch clamp. [Ca2+]i was measured by fura 2. PKC activity was measured by substrate phosphorylation after cell fractionation. PKC isoforms were assessed by Western blot and confocal microscopy. Nifedipine prevented the ischemia-induced increase in permeability in a dose-dependent manner. Ischemia increased [Ca2+]i, which was not affected by nifedipine. Instead, ischemia-induced PKC translocation was prevented by nifedipine. Phorbol ester also increased endothelial cell permeability, which was dose dependently inhibited by nifedipine. The effects of non-calcium-channel-binding dihydropyridine derivatives were similar. Analysis of the PKC isoforms showed that nifedipine prevented ischemia-induced translocation of PKC-alpha and PKC-zeta. Specific inhibition of PKC isoforms with antisense oligodeoxynucleotides demonstrated a major role for PKC-alpha. CONCLUSIONS: Nifedipine exerts a direct effect on endothelial cell permeability that is independent of calcium channels. The inhibition of ischemia-induced permeability by nifedipine seems to be mediated primarily by PKC-alpha inhibition. Anti-ischemic effects of dihydropyridine calcium antagonists could be due in part to their effects on endothelial cell permeability. (+info)
Paracrine role of adventitial superoxide anion in mediating spontaneous tone of the isolated rat aorta in angiotensin II-induced hypertension.
(27/2286)
The relationship between vascular generation of superoxide anion and spontaneous tone observed in the isolated aorta was studied in hypertensive rats infused with angiotensin II. Aortic rings from hypertensive, but not from sham-operated rats, demonstrated oscillatory spontaneous tone that represented 52+/-5.6% of the maximal contraction to KCl. Spontaneous tone was prevented by calcium-free buffer or by blocking calcium influx through L-type calcium channels with nifedipine. The production of superoxide anion measured by lucigenin chemiluminescence was up to 15-fold higher than in sham-operated rat aorta. The adventitial site of production of superoxide anion was suggested by the fact that lucigenin chemiluminescence was 5.5-fold higher from the adventitia than from the intima. This was confirmed histochemically by demonstrating that the adventitia was the site of reduction of nitroblue tetrazolium as well as immunohistochemical staining of NAD(P)H oxidase subunit proteins. A causal link between superoxide anion production by NAD(P)H oxidase and the spontaneous tone is suggested by the fact that superoxide dismutase or the inhibitor of NAD(P)H oxidase, diphenylene iodonium, decreased both superoxide anion production and spontaneous tone. L-NAME or removal of the endothelium from the aorta had no significant effect on superoxide anion levels or spontaneous tone. However, although superoxide dismutase decreased superoxide anion levels in the presence of L-NAME or in endothelium-denuded rings, it no longer inhibited the tone. This suggests that the effect on tone of superoxide anion originating in the adventitia is mediated by inactivating endothelium-derived nitric oxide, which promotes smooth muscle calcium influx and spontaneous tone. The adventitia is not a passive bystander during the development of hypertension, but rather it may have an important role in the regulation of smooth muscle tone. (+info)
Synergism between neuropeptide Y and norepinephrine highlights sympathetic cotransmission: studies in rat arterial mesenteric bed with neuropeptide Y, analogs, and BIBP 3226.
(28/2286)
Although abundant literature supports the notion that neuropeptide Y (NPY) synergizes in vivo and in vitro, the vasomotor activity elicited by norepinephrine (NE), the converse interaction (i.e., the adrenergic modulation of the NPY vasomotor response) has been less characterized. To assess whether NE synergizes the vasomotor effect of NPY, the rat arterial mesenteric bed was chosen as a model experimental system. Mesenteries were precontracted with NE and few minutes later were perfused with exogenous NPY. Under these conditions, NPY contracted the arterial mesenteric bed with an EC50 value of 0.72 +/- 0.06 nM. NPY was unable to contract this vascular territory without an agonist-induced precontraction. Other agonists, such as endothelin-1, a synthetic analog of prostaglandin F2alpha, or 5-hydroxytryptamine, also were effective primers because in their presence, NPY was a potent vasoconstrictor. In contrast, mesenteries precontracted with KCl failed to evidence the NPY-induced rise in perfusion pressure. Two structural analogs of NPY, PYY and [Leu31, Pro34]NPY, mimicked the activity of NPY. The NPY fragment 13-36 did not elicit such a response. All NPY analogs exhibited less efficacy and potency relative to NPY. The NPY- and related structural analog-induced vasoconstriction was competitively and reversibly antagonized by BIBP 3226; the pA2 of the NPY interaction was 7.0. The application of 0.1 to 1 microM BIBP 3226 or 0.1 to 10 nM prazosin at the peak of the NPY vasomotor response elicited a gradual blockade of the vasoconstriction. Although BIBP 3226 blocked the increase in perfusion pressure elicited by NPY, leaving unaffected the NE-induced tone, 10 nM prazosin blocked the full response, including the NE-induced component. Tissue preincubation with 200 nM nifedipine abolished the NPY-induced vasoconstriction; likewise, the acute application of 10 to 100 nM nifedipine blocked gradually the maximal NPY-induced contraction. Removal of the mesenteric endothelial layer increased the potency of NPY by 2-fold; it also slightly potentiated the antagonist activity of BIBP 3226. The synergism between NPY and NE backs the principle of sympathetic cotransmission. (+info)
Peptidyl inhibitors of shaker-type Kv1 channels elicit twitches in guinea pig ileum by blocking kv1.1 at enteric nervous system and enhancing acetylcholine release.
(29/2286)
Potent and selective peptidyl blockers of the Shaker-type (Kv1) voltage-gated potassium channels were used to determine the role of these channels in regulating the spontaneous motility of smooth muscle preparations. Margatoxin (MgTX), kaliotoxin, and agitoxin-2 at 1 to 10 nM and agitoxin-1 at 50 to 100 nM induce twitches in guinea pig ileum strips. These twitches are abolished by tetrodotoxin (TTX, 0.5 microM), atropine (1 microM), hexamethonium (10 microM), or nifedipine (0.1 microM). It is proposed that blockade of Kv1 channels by MgTX, kaliotoxin, or the agitoxins increases excitability of intramural nerve plexuses in the ileum, promoting release of acetylcholine from excitatory motor nerve terminals. This, in turn, leads to Ca2+-dependent action potentials and twitching of the muscle fibers. MgTX does not induce twitches in several other guinea pig and/or rat vascular, genitourinary, or gastrointestinal smooth muscles, although small increases in spontaneous myogenic activity may be seen in detrusor muscle exposed to >30 nM MgTX. This effect is not reversed by TTX or atropine. The TTX- and atropine-sensitive twitches of guinea pig ileum are also induced by nanomolar concentrations of alpha-dendrotoxin, a selective blocker of Shaker Kv1.1 and 1.2 subtypes, or stichodactylatoxin, a peptide isolated from sea anemone that displays high affinity for Kv1.1 and 1.3, but not by charybdotoxin, which blocks Kv1.2 and 1.3 but not 1.1. The data taken together suggest that high-affinity blockade of Kv1.1 underlies the ability of MgTX, kaliotoxin, agitoxin-1, agitoxin-2, alpha-dendrotoxin, and stichodactylatoxin to elicit TTX-sensitive twitches in guinea pig ileum. (+info)
In vivo measurement by [3H]Tamsulosin of alpha1 adrenoceptors in rat tissues in relation to the pharmacokinetics.
(30/2286)
The present study was undertaken to simultaneously measure alpha1 adrenoceptors in rat tissues by [3H]tamsulosin in vivo. In vivo specific [3H]tamsulosin binding was observed in the prostate, vas deferens, aorta, submaxillary gland, spleen, heart, lung, and kidney after i.v. injection of the ligand but not in the cerebral cortex and liver. Specific [3H]tamsulosin binding in the kidney, lung, heart, and spleen was greatest at 3 min after i.v. injection and declined rapidly with the disappearance of [3H]tamsulosin from the plasma. On the other hand, [3H]tamsulosin binding in the prostate and aorta peaked at 10 to 60 min after i.v. injection, and a considerable level of specific binding in both tissues persisted up to 240 min. The most sustained binding of [3H]tamsulosin occurred in the submaxillary gland. In vivo specific [3H]tamsulosin binding in rat tissues was effectively inhibited by the coinjection of low doses of unlabeled tamsulosin, prazosin, and terazosin with the radioligand but not by relatively high doses of yohimbine and propranolol. Based on estimated ID50 values, in vivo inhibitory effect of tamsulosin compared with prazosin was 5 to 14 times greater in rat tissues except the spleen, which showed 1.6 times less potent than prazosin. From ratios of ID50 (spleen) to ID50 (submaxillary gland) or ID50 (prostate), tamsulosin was 9 and 19 times, respectively, greater than prazosin in selectivity of alpha1 adrenoceptors in the submaxillary gland and prostate versus the spleen, respectively, suggesting that tamsulosin binds to alpha1A subtype with higher affinity than alpha1B subtype in vivo. The present study suggests that [3H]tamsulosin is a useful radioligand for in vivo measurement of alpha1 adrenoceptors in rat tissues. (+info)
Role of protein kinase C in mitochondrial KATP channel-mediated protection against Ca2+ overload injury in rat myocardium.
(31/2286)
Growing evidence exists that ATP-sensitive mitochondrial potassium channels (MitoKATP channel) are a major contributor to the cardiac protection against ischemia. Given the importance of mitochondria in the cardiac cell, we tested whether the potent and specific opener of the MitoKATP channel diazoxide attenuates the lethal injury associated with Ca2+overload. The specific aims of this study were to test whether protection by diazoxide is mediated by MitoKATP channels; whether diazoxide mimics the effects of Ca2+ preconditioning; and whether diazoxide reduces Ca2+ paradox (PD) injury via protein kinase C (PKC) signaling pathways. Langendorff-perfused rat hearts were subjected to the Ca2+ PD (10 minutes of Ca2+ depletion followed by 10 minutes of Ca2+ repletion). The effects of the MitoKATP channel and other interventions on functional, biochemical, and pathological changes in hearts subjected to Ca2+ PD were assessed. In hearts treated with 80 micromol/L diazoxide, left ventricular end-diastolic pressure and coronary flow were significantly preserved after Ca2+ PD; peak lactate dehydrogenase release was also significantly decreased, although ATP content was less depleted. The cellular structures were well preserved, including mitochondria and intercalated disks in diazoxide-treated hearts compared with nontreated Ca2+ PD hearts. The salutary effects of diazoxide on the Ca2+ PD injury were similar to those in hearts that underwent Ca2+ preconditioning or pretreatment with phorbol 12-myristate 13-acetate before Ca2+ PD. The addition of sodium 5-hydroxydecanoate, a specific MitoKATP channel inhibitor, or chelerythrine chloride, a PKC inhibitor, during diazoxide pretreatment completely abolished the beneficial effects of diazoxide on the Ca2+ PD. Blockade of Ca2+ entry during diazoxide treatment by inhibiting L-type Ca2+ channel with verapamil or nifedipine also completely reversed the beneficial effects of diazoxide on the Ca2+ PD. PKC-delta was translocated to the mitochondria, intercalated disks, and nuclei of myocytes in diazoxide-pretreated hearts, and PKC-alpha and PKC-epsilon were translocated to sarcolemma and intercalated disks, respectively. This study suggests that the effect of the MitoKATP channel is mediated by PKC-mediated signaling pathway. (+info)
BACKGROUND: It has been suggested that intracellular Ca2+ overload in cardiac myocytes leads to the development of diabetic cardiomyopathy. Troglitazone, an insulin-sensitizing agent, is a promising therapeutic agent for diabetes and has been shown to prevent diabetes-induced myocardial changes. To elucidate the underlying mechanism of troglitazone action on cardiac myocytes, the effects of troglitazone on voltage-dependent Ca2+ currents were examined and compared with classic Ca2+ antagonists (verapamil and nifedipine). METHODS AND RESULTS: Whole-cell voltage-clamp techniques were applied in single guinea pig atrial myocytes. Under control conditions with CsCl internal solution, the voltage-dependent Ca2+ currents consisted of both T-type (ICa,T) and L-type (ICa,L) Ca2+ currents. Troglitazone effectively reduced the amplitude of ICa,L in a concentration-dependent manner. Troglitazone also suppressed ICa,T, but the effect of troglitazone on ICa,T was less potent than that on ICa,L. The current-voltage relationships for ICa,L and the reversal potential for ICa,L were not altered by troglitazone. The half-maximal inhibitory concentration of troglitazone on ICa,L measured at a holding potential of -40 mV was 6.3 micromol/L, and 30 micromol/L troglitazone almost completely inhibited ICa,L. Troglitazone 10 micromol/L did not affect the time courses for inactivation of ICa,L and inhibited ICa,L mainly in a use-independent fashion, without shifting the voltage-dependency of inactivation. This effect was different from those of verapamil and nifedipine. Troglitazone also reduced isoproterenol- or cAMP-enhanced ICa,L. CONCLUSIONS: These results demonstrate that troglitazone inhibits voltage-dependent Ca2+ currents (T-type and L-type) and then antagonizes the effects of isoproterenol in cardiac myocytes, thus possibly playing a role in preventing diabetes-induced intracellular Ca2+ overload and subsequent myocardial changes. (+info)