Acute and chronic effects of capsaicin in perfused rat muscle: the role of tachykinins and calcitonin gene-related peptide

In perfused rat skeletal muscle (hindlimb), capsaicin either stimulates (submicromolar concentrations) or inhibits (micromolar concentrations) oxygen consumption (VO2). Both VO2 effects are associated with vasoconstriction, evident as an increase in perfusion pressure (PP), under constant flow. We have proposed that these effects are mediated by two vanilloid receptor subtypes: VN1 (stimulation of VO2) and VN2 (inhibition of VO2) (; ). In the present study, the role of capsaicin-sensitive neurons and sensory neuropeptides in the VN1/VN2 receptor actions of capsaicin was investigated. The observed maximum stimulation of VO2 by capsaicin (0.4 microM; DeltaVO2, 1.35 +/- 0.14 micromol g-1 h-1) was accompanied by mild vasoconstriction (DeltaPP, 5.8 +/- 0.6 mm Hg). In contrast, 2 microM capsaicin produced strong inhibition of VO2 (DeltaVO2, -2.25 +/- 0.23 micromol g-1 h-1) with pronounced vasoconstriction (DeltaPP, 28.0 +/- 1.3 mm Hg). VO2 stimulation was significantly inhibited (P <.05) by the selective NK1 receptor antagonist CP-99994 (1 microM) and the NK2 receptor antagonist SR 48968 (1 microM) (by 42% and 51%, respectively), but PP was not altered. Infused SP and neurokinin A (NKA) stimulated VO2 (observed maximum DeltaVO2, 0.52 +/- 0.06 and 0.53 +/- 0.08 micromol g-1 h-1, respectively; EC50 values, 269 +/- 23 and 21.2 +/- 3.0 nM, respectively) and induced mild vasoconstriction (4.30 +/- 0.33 and 6. 75 +/- 1.18 mm Hg, respectively; EC50 values, 352 +/- 25.7 and 25.5 +/- 2.7 nM, respectively). Neurokinin B (NKB) also stimulated VO2 (maximum not determined) and vasoconstriction (maximum DeltaPP, 3.40 +/- 0.25 mm Hg; EC50, 34.4 +/- 5.2 nM). The rank order of potency for the tachykinins in this preparation was NKA > NKB > SP, which suggests stimulation primarily of NK2 receptors. Although infused calcitonin gene-related peptide (CGRP) did not alter hindlimb VO2 or PP, the selective CGRP antagonist CGRP(8-37) markedly potentiated the inhibition of VO2 produced by 1 microM capsaicin (84%) and the maximum capsaicin-induced vasoconstriction (57%), which indicates that endogenously released CGRP may act as a vasodilator. Hindlimbs perfused 1 day after capsaicin pretreatment showed attenuation of capsaicin-induced (0.4 microM) stimulation of VO2 (92%) (P <.05) and vasoconstriction (64%), but this returned to normal after 7 days. The inhibition of VO2 by 1 microM capsaicin was significantly (P <. 05) enhanced 7 and 14 days after pretreatment (66% and 140%, respectively), as was the maximum vasoconstriction (64% and 68%, respectively). These data suggest that capsaicin-sensitive neurons, presumably via release of SP and NKA, are involved in VN1 responses and that capsaicin pretreatment potentiates VN2 responses, either by depletion of CGRP reserves or by upregulation of putative VN2 receptors.     

Role of K+ channel opening and stimulation of cyclic GMP in the vasorelaxant effects of nicorandil in isolated piglet pulmonary and mesenteric arteries: relative efficacy and interactions between both pathways

1. The effects of the K+ channel opener levcromakalim, the guanylate cyclase stimulant nitroprusside and the dual drug nicorandil (K+ channel opener and guanylate cyclase stimulant) were analysed in piglet isolated endothelium-denuded pulmonary (PA) and mesenteric (MA) arteries stimulated by noradrenaline (NA) or by the thromboxane A2 mimetic U46619. 2. Nicorandil, levcromakalim and verapamil were less potent in PA than in MA, the efficacy of levcromakalim was also reduced in PA. The effects of nicorandil and levcromakalim were similar in arteries pre-contracted by NA and U46619, whereas verapamil was more potent in arteries pre-contracted by NA. Nitroprusside was equipotent in MA pre-contracted by either NA or U46619 and in PA pre-contracted by NA whereas in PA pre-contracted by U46619, nitroprusside showed lower potency and efficacy. 3. The relaxant effects of levcromakalim and nitroprusside were inhibited by 10(-5) M glibenclamide and 10(-6) M ODQ, respectively. Nicorandil-induced relaxation was inhibited by ODQ in all experimental conditions, whereas glibenclamide had inhibitory effects in PA and MA pre-contracted by U46619, had no effect in PA pre-contracted by NA and in MA pre-contracted by NA it was only inhibitory in the presence of ODQ. 4. No apparent interactions were found between nitroprusside and levcromakalim as indicated by the lack of effects of pretreatment with one of them (producing 20-35% relaxation) on the potency of the relaxant response to the other. However, in PA pre-contracted by U46619, where nitroprusside or levcromakalim induced only partial relaxation, the combination of both mechanisms (either by combining nitroprusside plus levcromakalim or by nicorandil) was able to induce full vasodilatation. 5. In conclusion, K+ channel opening and guanylate cyclase stimulation are independent pathways that induce additive vasorelaxation in piglet PA and MA. The mechanism of action of nicorandil is dependent on the artery and on the nature of the agonist employed to precontract the artery. The relative efficacy of K+ channel opening vs guanylate cyclase stimulation may partially explain the preferential contribution of each mechanism to the relaxant effects of nicorandil.     

Effects of calcitonin gene-related peptide on wound contraction in denervated and normal rat skin: a preliminary report

The aim of the present study was to observe the effect of calcitonin gene-related peptide (CGRP) on wound contraction in both denervated and normal areas. A total of 100 Wistar rats, each of which had a 2 x 2 cm full-thickness skin defect on the back, were divided into two main control groups and six corresponding experimental groups in which 10 x 10(-9) M and 10 x 10(-12) M synthetic rat CGRP were given intraperitoneally and intradermally. Contraction was assessed weekly with planimetry, and mean surface areas (mm2 +/- SD) of related groups were compared. Complete closure took 4 weeks for the normal control group and 8 weeks for the denervated control group (p < 0.05). The 10 x 10(-12) M CGRP with both types of application showed a decrease in the length of time for complete closure to 3 weeks in the normal experimental groups (p < 0.05), but complete closure still took 4 weeks in normal groups in which 10 x 10(-9) M CGRP was given (p < 0.05). Any dosage of CGRP given intraperitoneally showed no change in the closure period in the denervated experimental groups (p > 0.05). CGRP showed a trophic effect on healing by an increased rate of contraction in the rat model. However, the neural supply to the wound area seemed to be intact because of the necessity of axonal transfer of CGRP.     

Noxiustoxin 2, a novel K+ channel blocking peptide from the venom of the scorpion Centruroides noxius Hoffmann

A novel peptide called Noxiustoxin 2 (NTX2) was purified from the venom of the scorpion Centruroides noxius and characterized chemically and functionally. It is composed of 38 amino acid residues linked by three disulfide bridges and its primary structure is 61% identical to that of Noxiustoxin (NTX). It is not toxic to mice (using up to 200 micrograms/20 g mouse weight) and crustaceans (up to 30 micrograms/g of crayfish), but has a paralysing effect on crickets (30 micrograms/g animal). It displaces the binding of [125I]NTX to rat brain synaptosome membranes with a Ki of 0.1 microM, in comparison NTX has a Ki of 100 pM. Similarly, using single Ca2+ activated K+ channels of small conductance obtained from cultured bovine aortic endothelial cells it was shown that NTX2 is over two logarithm units less potent than NTX in producing 50% blockade of the probability of opening the channels. NTX2 is not recognized by a panel of six distinct monoclonal antibodies against NTX, however it is recognized by polyclonal antibodies raised in mouse, with native NTX. Primary structure comparison of both NTX and NTX2 suggests that the N-terminal segments of these peptides are important for channel affinity.     

Class III antiarrhythmic drugs block HERG, a human cardiac delayed rectifier K+ channel. Open-channel block by methanesulfonanilides

We recently reported that mutations in HERG, a potassium channel gene, cause long QT syndrome. Heterologous expression of HERG in Xenopus oocytes revealed that this channel had biophysical properties nearly identical to a cardiac delayed rectifier K+ current I(Kr), but had dissimilar pharmacological properties. Class III antiarrhythmic drugs such as E-4031 and MK-499 are potent and specific blockers of I (Kr) in cardiac myocytes. Our initial studies indicated that these compounds did not block HERG at a concentration of 1 micromol/L. In the present study, we used standard two-microelectrode voltage-clamp techniques to further characterize the effects of these drugs on HERG channels expressed in oocytes. Consistent with initial findings, 1 micromol/L MK-499 and E-4031 had not effect on HERG when oocytes were voltage clamped at a negative potential and not pulsed during equilibration with the drug. However, MK-499 did block HERG current if oocytes were repetitively pulsed, or clamped at a voltage positive to the threshold potential for channel activation. This finding is in contrast to previous studies that showed significant block of I(Kr) in isolated myocytes by similar drugs, even in the absence of pulsing. This apparent discrepancy may be due to differences in channel characteristics (HERG versus guinea pig and mouse I (Kr)), tissue (oocytes versus myocytes), or specific drugs. Under steady state conditions, block of HERG by MK-499 was half maximal at 123 +/- 12 nmol/L at a test potential of -20 mV. MK-499 (150 nmol/L) did not affect the voltage dependence of activation and rectification nor the kinetics of activation and deactivation of HERG. These data indicate that MK-499 preferentially blocks open HERG channels and further support the conclusion that HERG subunits form I(Kr) channels in cardiac myocytes.     

Expression environment determines K+ current properties: Kv1 and Kv4 alpha-subunit-induced K+ currents in mammalian cell lines and cardiac myocytes

Voltage-gated K+ channel (Kv) pore-forming (alpha) subunits of the Kv1 and Kv4 subfamilies have been cloned from heart cDNA libraries, and are thought to play roles in the generation of the transient outward K+ current, Ito. Heterologous expression of these subunits in Xenopus oocytes, however, reveals K+ currents that are quite distinct from Ito. In the experiments here, the detailed time- and voltage-dependent properties of the currents expressed in mammalian cell lines and in cardiac myocytes by Kv1.4 and Kv4.2 were examined and compared to previous findings in studies of oocytes, as well as to Ito characterized in various myocardial cells. As in oocytes, expression of Kv1.4 in HEK-293, Ltk- or neonatal rat ventricular cells reveals rapidly activating K+ currents. In contrast to the currents in oocytes, however, there are two components of inactivation of the Kv1.4-induced currents in mammalian cells, and both components are significantly slower in myocytes than in either HEK-293 or Ltk- cells. In addition, in all three cell types, recovery of Kv1.4 from steady-state inactivation is very slow, proceeding with mean time constants in the range of 6-8 s. The properties of Kv4.2-induced currents also vary with cell type and, importantly, the rates of activation, inactivation and recovery from inactivation are significantly faster in mammalian cells than in Xenopus oocytes. In HEK-293, Chinese hamster ovary (CHO) and neonatal rat ventricular cells, for example, the currents recover from steady-state inactivation with mean (+/-SD) time constants of 153+/-32 (n=12), 245+/-112 (n=10) and 86+/-38 (n=11) ms, respectively; therefore, recovery proceeds 5-10 times faster than observed for Kv4.2 in oocytes. These results emphasize the importance of the cellular expression environment in efforts to correlate endogenous K+ currents with heterologously expressed K+ channel subunits. In addition, the finding that Kv alpha subunits produce distinct K+ currents in different cells suggests that cell-type-specific associations with endogenous Kv alpha or accessory beta subunits and/or post-translational processing play roles in determining the properties of functional K+ channels.     

Extracellular ATP and UTP control the generation of reactive oxygen intermediates in human macrophages through the opening of a charybdotoxin-sensitive Ca2+-dependent K+ channel

Human monocyte-derived macrophages possess a NADPH oxidase that catalyzes superoxide formation upon phagocytosis. Extracellular ATP per se does not activate NADPH oxidase but potentiates superoxide generation triggered by opsonized zymosan. UTP can substitute for ATP with the same efficiency, suggesting that ATP mediates its effects specifically through P2U receptors. Extracellular UTP stimulates a rapid increase in cytoplasmic Ca2+ concentration in monocytic cells, which results from a release of intracellular Ca2+ stores. Moreover, UTP-induced calcium increase is sufficient to activate a charybdotoxin-sensitive Ca2+-dependent outward K+ channel (K(Ca)). The activity of this channel develops between 0.1 and 1.0 microM free cytoplasmic Ca2+ concentration; it is half-blocked by 10 nM charybdotoxin but insensitive to iberiotoxin. Under asymmetrical K+ conditions, this K(Ca) channel does not depend on membrane potential and is characterized by a linear single-current voltage relationship in the voltage range of -100 to +50 mV, giving a unitary conductance of 10 pico-Siemens. Interestingly, ATP/UTP-induced oxygen radicals release was inhibited by charybdotoxin in the same range of concentration as the UTP-induced K(Ca) channel. Furthermore, we show that ATP or UTP fail to enhance oxygen radicals production before K(Ca) channel is expressed (3 days). The electrogenic nature of the NADPH oxidase, i.e., its level of activation, being dependent on the plasmic membrane potential, might provide the causal link between the reactive oxygen intermediates generation and the opening of the K(Ca) channel.     

Time domains of neuronal Ca2+ signaling and associative memory: steps through a calexcitin, ryanodine receptor, K+ channel cascade

Synaptic changes that underlie associative learning and memory begin with temporally related activity of two or more independent synaptic inputs to common postsynaptic targets. In turn, temporally related molecular events regulate cytosolic Ca2+ during progressively longer-lasting time domains. Associative learning behaviors of living animals have been correlated with changes of neuronal voltage-dependent K+ currents, protein kinase C-mediated phosphorylation and synthesis of the Ca2+ and GTP-binding protein, calexcitin (CE),and increased expression of the Ca2+-releasing ryanodine receptor (type II). These molecular events, some of which have been found to be dysfunctional in Alzheimer's disease, provide means of altering dendritic excitability and thus synaptic efficacy during induction, consolidation and storage of associative memory. Apparently, such stages of behavioral learning correspond to sequential differences of Ca2+ signaling that could occur in spatially segregated dendritic compartments distributed across brain structures, such as the hippocampus.     

Structure-activity relationship of a novel K+ channel opener, KRN4884, and related compounds in porcine coronary artery

1. KRN4884 (5-amino-N-[2-(2-chlorophenyl) ethyl]-N'-cyano-3-pyridinecarboxamidine), Ki3005 (5-deamino KRN4884), Ki5624 (2-dechloro KRN4884) and Ki1769 (5-deamino-2-dechloro KRN4884) produced concentration-dependent relaxations in isolated porcine coronary arteries contracted by 25 mM KC1. The order of relaxant potency was KRN4884 > Ki3005 > Ki5624 > Ki1769. 2. The relaxation induced by these compounds was antagonized by glibenclamide; they had almost no effect on coronary arteries contracted by 60 mM KC1. 3. The present results suggest that these pyridinecarboxamidine derivatives have vasodilating ability based on a K+ channel opening action, and that both the amino groups in the pyridine nucleus and the chlorine atom in the benzene nucleus in pyridinecarboxamidine are important for their potency as a K+ channel opener.     

Effects of a K+ATP channel opener, lemakalim, on systemic, coronary and regional vascular dynamics in conscious dogs: comparison with nifedipine, adenosine, nitroglycerin and acetylcholine

The systemic, coronary and regional vascular responses to the K+ATP channel opener lemakalim were compared to other potent vasodilators (i.e., nifedipine, adenosine, nitroglycerin and acetylcholine). Experiments were performed in 12 conscious dogs 2 to 4 weeks after implantation of aortic catheters and flow probes on the ascending aorta, left circumflex coronary, celiac, mesenteric, renal and iliac arteries, and solid-state miniature pressure gauges in the left ventricular cavity. Dose-response curves induced by bolus injection (i.v.) were examined. For doses that reduced total peripheral resistance by 22%, lemakalim reduced celiac (-28 +/- 2%), mesenteric (-24 +/- 3%), renal (-17 +/- 3%) and iliac (-18 +/- 3%) vascular resistances (i.e., by amounts similar to those observed with the other vasodilators, except for adenosine, which increased renal resistance). At these doses, lemakalim induced a greater decrease (-52 +/- 3%) (P < .05) in coronary resistance, as compared with nifedipine (-35 +/- 3%), adenosine (-38 +/- 3%), nitroglycerin (-25 +/- 2%) and acetylcholine (-32 +/- 3%). However, when near maximal vasodilation was elicited, adenosine elicited the greatest (P < .05) decrease in coronary resistance (-81 +/- 1%), as compared with lemakalim (-74 +/- 2%), nifedipine (-67 +/- 2%), nitroglycerin (-63 +/- 2%) and acetylcholine (-72 +/- 1%). Both the time to maximal increases in regional blood flow and the time for recovery in all vascular beds were significantly prolonged for lemakalim compared with the other vasodilators. Thus, the K+ATP channel opener lemakalim dilates the coronary bed out of proportion to other vascular beds, is relatively more potent at lower doses than other vasodilators and exhibits a delayed and more prolonged action in all regional vascular beds.     

The large conductance, voltage-dependent, and calcium-sensitive K+ channel, Hslo, is a target of cGMP-dependent protein kinase phosphorylation in vivo

Native large conductance, voltage-dependent, and Ca2+-sensitive K+ channels are activated by cGMP-dependent protein kinase. Two possible mechanisms of kinase action have been proposed: 1) direct phosphorylation of the channel and 2) indirect via PKG-dependent activation of a phosphatase. To scrutinize the first possibility, at the molecular level, we used the human pore-forming alpha-subunit of the Ca2+-sensitive K+ channel, Hslo, and the alpha-isoform of cGMP-dependent protein kinase I. In cell-attached patches of oocytes co-expressing the Hslo channel and the kinase, 8-Br-cGMP significantly increased the macroscopic currents. This increase in current was due to an increase in the channel voltage sensitivity by approximately 20 mV and was reversed by alkaline phosphatase treatment after patch excision. In inside-out patches, however, the effect of purified kinase was negative in 12 of 13 patches. In contrast, and consistent with the intact cell experiments, purified kinase applied to the cytoplasmic side of reconstituted channels increased their open probability. This stimulatory effect was absent when heat-denatured kinase was used. Biochemical experiments show that the purified kinase incorporates gamma-33P into the immunopurified Hslo band of approximately 125 kDa. Furthermore, in vivo phosphorylation largely attenuates this labeling in back-phosphorylation experiments. These results demonstrate that the alpha-subunit of large conductance Ca2+-sensitive K+ channels is substrate for G-Ialpha kinase in vivo and support direct phosphorylation as a mechanism for PKG-Ialpha-induced activation of maxi-K channels.     

New structure and function in plant K+ channels: KCO1, an outward rectifier with a steep Ca2+ dependency

Potassium (K+) channels mediating important physiological functions are characterized by a common pore-forming (P) domain. We report the cloning and functional analysis of the first higher plant outward rectifying K+ channel (KCO1) from Arabidopsis thaliana. KCO1 belongs to a new class of 'two-pore' K+ channels recently described in human and yeast. KCO1 has four putative transmembrane segments and tandem calcium-binding EF-hand motifs. Heterologous expression of KCO1 in baculovirus-infected insect (Spodoptera frugiperda) cells resulted in outwardly rectifying, K+-selective currents elicited by depolarizing voltage pulses in whole-cell measurements. Activation of KCO1 was strongly dependent on the presence of nanomolar concentrations of cytosolic free Ca2+ [Ca2+]cyt. No K+ currents were detected when [Ca2+]cyt was adjusted to <150 nM. However, KCO1 strongly activated at increasing [Ca2+]cyt, with a saturating activity observed at approximately 300 nM [Ca2+]cyt. KCO1 single channel analysis on excised membrane patches, resulting in a single channel conductance of 64 pS, confirmed outward rectification as well as Ca2+-dependent activation. These data suggest a direct link between calcium-mediated signaling processes and K+ ion transport in higher plants. The identification of KCO1 as the first plant K+ outward channel opens a new field of structure-function studies in plant ion channels.     

Identification of the reactive histidine of cucumisin, a plant serine protease: modification with peptidyl chloromethyl ketone derivative of peptide substrate

A radioactive peptidyl chloromethyl ketone derived from substrates of cucumisin, 3H-labeled Z-Ala-Ala-Pro-Phe-chloromethyl ketone (3H-ZAAPFCK), was synthesized. When cucumisin was incubated with a 100-fold molar excess of 3H-ZAAPFCK for 16 h, 98% of the cucumisin activity was inhibited and about 0.93 mol of 3H-ZAAPFCK was incorporated in 1 mol of cucumisin. The 3H-ZAAPFCK-modified cucumisin was reduced and pyridylethylated, and then digested by trypsin. The radioactive peptide fragment was isolated and its amino acid sequence was determined. The radioactive fragment contained 32 amino acid residues and the sequence around the labeled residue was found to be -Asp-Thr-Asn-Gly-(His)-Gly-Thr-His-Thr-Ala-. This sequence is analogous to that around the reactive site histidine residue of the subtilisin family.     

Bimoclomol: a nontoxic, hydroxylamine derivative with stress protein-inducing activity and cytoprotective effects

Preservation of the chemical architecture of a cell or of an organism under changing and perhaps stressful conditions is termed homeostasis. An integral feature of homeostasis is the rapid expression of genes whose products are specifically dedicated to protect cellular functions against stress. One of the best known mechanisms protecting cells from various stresses is the heat-shock response which results in the induction of the synthesis of heat-shock proteins (HSPs or stress proteins). A large body of information supports that stress proteins--many of them molecular chaperones--are crucial for the maintenance of cell integrity during normal growth as well as during pathophysiological conditions, and thus can be considered "homeostatic proteins." Recently emphasis is being placed on the potential use of these proteins in preventing and/or treating diseases. Therefore, it would be of great therapeutic benefit to discover compounds that are clinically safe yet able to induce the accumulation of HSPs in patients with chronic disorders such as diabetes mellitus, heart disease or kidney failure. Here we show that a novel cytoprotective hydroxylamine derivative, [2-hydroxy-3-(1-piperidinyl) propoxy]-3-pyridinecarboximidoil-chloride maleate, Bimoclomol, facilitates the formation of chaperone molecules in eukaryotic cells by inducing or amplifying expression of heat-shock genes. The cytoprotective effects observed under several experimental conditions, including a murine model of ischemia and wound healing in the diabetic rat, are likely mediated by the coordinate expression of all major HSPs. This nontoxic drug, which is under Phase II clinical trials, has enormous potential therapeutic applications.     

Wanderlust kinetics and variable Ca(2+)-sensitivity of Drosophila, a large conductance Ca(2+)-activated K+ channel, expressed in oocytes

Cloned large conductance Ca(2+)-activated K+ channels (BK or maxi-K+ channels) from Drosophila (dSlo) were expressed in Xenopus oocytes and studied in excised membrane patches with the patch-clamp technique. Both a natural variant and a mutant that eliminated a putative cyclic AMP-dependent protein kinase phosphorylation site exhibited large, slow fluctuations in open probability with time. These fluctuations, termed "wanderlust kinetics," occurred with a time course of tens of seconds to minutes and had kinetic properties inconsistent with simple gating models. Wanderlust kinetics was still observed in the presence of 5 mM caffeine or 50 nM thapsigargin, or when the Ca2+ buffering capacity of the solution was increased by the addition of 5 mM HEDTA, suggesting that the wanderlust kinetics did not arise from Ca2+ release from caffeine and thapsigargin sensitive internal stores in the excised patch. The slow changes in kinetics associated with wanderlust kinetics could be generated with a discrete-state Markov model with transitions among three or more kinetic modes with different levels of open probability. To average out the wanderlust kinetics, large amounts of data were analyzed and demonstrated up to a threefold difference in the [Ca2+]i required for an open probability of 0.5 among channels expressed from the same injected mRNA. These findings indicate that cloned dSlo channels in excised patches from Xenopus oocytes can exhibit large variability in gating properties, both within a single channel and among channels.     

The effects of nucleotides and potassium channel openers on the SUR2A/Kir6.2 complex K+ channel expressed in a mammalian cell line, HEK293T cells

The aim of this study was to evaluate whether long-term administration of L-arginine, a physiological substrate for the production of nitric oxide, improved blood pressure, heart rate, cardiac hypertrophy and particularly structural changes in the coronary and carotid artery of spontaneously hypertensive rats (SHR). The experiments started with three groups of 10-week-old animals: control Wistar rats, untreated SHR and SHR treated with L-arginine (SHR + L-arginine). L-Arginine was administered to SHR in a daily dose of 300 mg kg-1 intraperitoneally for 6 weeks. Blood pressure and heart rate were recorded each week. At the end of the experiment in one-half of each group heart weight and body weight were determined and the heart weight/body weight index was calculated. In the other animals, the cardiovascular system was perfused via the left ventricle with a glutaraldehyde fixative at 120 mmHg and the coronary and carotid arteries were processed for transmission electron microscopy. The inner diameter and wall thickness (tunica intima and tunica media) were measured on semithin sections. The reliability of the genetic feature in the SHR group was proved by the increased heart weight, heart weight/body weight index, wall thickness and wall thickness/inner diameter ratio of coronary and carotid arteries in comparison to the group of control Wistar rats. Long-term administration of L-arginine did not significantly influence blood pressure and heart rate in comparison with untreated SHR. Neither were any differences found in cardiac hypertrophy or the geometry of the coronary and carotid arteries (thickness of arterial wall, inner diameter, wall/diameter ratio). In conclusion, the changes in the cardiovascular system in SHR were not reversed, or even alleviated, by chronic treatment with L-arginine.     

Synthesis, electrophysiological properties and analysis of structural requirements of a novel class of antiarrhythmic agents with potassium and calcium channel blocking properties

Class III antiarrhythmic agents have been shown to prevent reentrant arrhythmias but also to be responsible for initiating arrhythmias characterised by afterdepolarizations and triggered activities. By combining potassium and calcium channel antagonistic actions, as with BRL-32872 (1), it might be possible to reduce the incidence of proarrhythmias albeit retaining antiarrhythmic efficacy. In the present study we synthesised and tested for their electrophysiological activity in guinea pig papillary muscle a wide panel of analogues of BRL-32872. Some qualitative relationships between compound structure and the inhibitory effect on the rapidly activating component of the delayed rectifier potassium current and/or the L-type calcium current will be presented. New derivatives depicting bell-shaped dose-response curves on action potential duration may therefore represent novel agents for improved antiarrhythmic therapy.     

Kv8.1, a new neuronal potassium channel subunit with specific inhibitory properties towards Shab and Shaw channels

Outward rectifier K+ channels have a characteristic structure with six transmembrane segments and one pore region. A new member of this family of transmembrane proteins has been cloned and called Kv8.1. Kv8.1 is essentially present in the brain where it is located mainly in layers II, IV and VI of the cerebral cortex, in hippocampus, in CA1-CA4 pyramidal cell layer as well in granule cells of the dentate gyrus, in the granule cell layer and in the Purkinje cell layer of the cerebellum. The Kv8.1 gene is in the 8q22.3-8q24.1 region of the human genome. Although Kv8.1 has the hallmarks of functional subunits of outward rectifier K+ channels, injection of its cRNA in Xenopus oocytes does not produce K+ currents. However Kv8.1 abolishes the functional expression of members of the Kv2 and Kv3 subfamilies, suggesting that the functional role of Kv8.1 might be to inhibit the function of a particular class of outward rectifier K+ channel types. Immunoprecipitation studies have demonstrated that inhibition occurs by formation of heteropolymeric channels, and results obtained with Kv8.1 chimeras have indicated that association of Kv8.1 with other types of subunits is via its N-terminal domain.     

Inhibition by protein kinase C of the 86Rb+ efflux and vasorelaxation induced by P1075, a K(ATP) channel opener, in rat isolated aorta

The expression of GABA in the human fetal (12-25 weeks of gestation), postnatal (five-month-old), and adult (35-year-old) retinas was investigated by immunohistochemistry. GABA expression was seen as early as 12 weeks in the undifferentiated cells of the inner neuroblast zone; a few optic nerve fiber layer axons were clearly labeled, suggesting that some of the stained cell bodies were prospective ganglion cells, others could be displaced amacrine cells. From 16-17 to 24-25 weeks, intense labeling was found in the amacrine, displaced amacrine, and some ganglion cells. During this time period, horizontal cells (identified by calbindin immunohistochemistry), undergoing migration (periphery) and differentiation (center), expressed GABA prominently. In the postnatal retina, some horizontal cells were moderately labeled, but very weakly in a few cells, in the adult. The Müller cells developed immunoreactivity first weakly at 12 weeks and then moderately from 16-17 weeks onward. The staining was also evident in the postnatal and adult retinas, showing labeled processes of these glial cells. Virtually no axons in the adult optic nerve and nerve fiber layer were stained; the staining was restricted to a few, large ganglion cells and displaced amacrine cells: Some amacrines were also labeled. The possibility that GABA might play a role in horizontal cell differentiation and maturation is highlighted. Other evidences suggest that GABA might play a role in metabolism during retinal development.