Presence Of Blockers Research Articles

Role of Rho-associated protein kinase in tone and calcium sensitivity of cannulated rat mesenteric small arteries.

The regulation of vascular tone includes modulation of contractile element calcium sensitivity. We tested the involvement of the Rho-associated protein kinase p160ROCK in tone and calcium sensitivity of cannulated rat mesenteric small arteries. These vessels developed basal tone and showed myogenic responses upon pressure steps, resulting from an increase in calcium in combination with a high contractile element calcium sensitivity. Y-27632, believed to be a specific p160ROCK inhibitor, caused concentration-dependent inhibition of basal tone, with near full inhibition at 3 microM. At this concentration, myogenic responses were absent and stepwise pressure elevation resulted in severe vascular distension. Y-27632 did not affect pressure-induced changes in intracellular calcium but rather reduced pressure-induced as well as phenylephrine-induced calcium sensitisation. Thus in the presence of the blocker, for a given calcium concentration, tone was greatly reduced, and the divergence in sensitivity between pressure and phenylephrine as stimuli on the one hand and potassium on the other disappeared. K+ (125 mM) and ionomycin still caused contraction in the presence of the p160ROCK blocker. These data show that in pressurised small arteries the Rho-p160ROCK pathway is active in the absence of vasoconstrictors, keeping the vessels in a state of high calcium sensitivity and basal tone.

Reflex-elicited increases in female rat parotid protein synthesis involving parasympathetic non-adrenergic, non-cholinergic mechanisms.

Normal rats or rats parasympathetically denervated on one side by cutting the auriculo-temporal nerve were maintained on a liquid diet for 1 week. After fasting overnight, experimental rats were then offered hard, pelleted food over a period of 2 h, while unfed animals served as controls. The reflex-induced increase in the [3H]leucine incorporation into trichloroacetic acid-insoluble material of the parotid glands following intake of the food in the presence of the muscarinic blocker atropine and alpha- and beta-adrenoceptor antagonists was greater (104%) than in the absence of the blockers (75%) in normal rats. The picture was the same when the innervated glands of rats subjected to unilateral parasympathetic denervation were examined. In this case, the protein synthesis increased by 108% in the presence of the blockers and by 63% in their absence. Analysis of the parasympathetically denervated and contralateral innervated glands revealed no support for a cholinergic or adrenergic contribution to this response. The increase in protein synthesis in the innervated glands was 86% in the rats treated with atropine, whilst it was 76% in those treated with alpha- and beta-adrenoceptor antagonists. The protein synthesis in the denervated glands increased by 57% in the absence of receptor blockade. Neither atropine (51%), nor a- and b-adrenoceptor antagonists (52%), nor a combination of the three blockers (55%) affected the response of the denervated glands. A large part of the response in the presence of the traditional autonomic receptor blockers was thus dependent on the parasympathetic auriculo-temporal nerve. Under natural feeding conditions, the parasympathetic non-adrenergic, non-cholinergic (NANC) mechanisms are likely to contribute to the synthesis of secretory proteins.

Passive water and ion transport by cotransporters.

1. The rabbit Na+-glucose (SGLT1) and the human Na+-Cl--GABA (GAT1) cotransporters were expressed in Xenopus laevis oocytes, and passive Na+ and water transport were studied using electrical and optical techniques. Passive water permeabilities (Lp) of the cotransporters were determined from the changes in oocyte volume in response to osmotic gradients. The specific SGLT1 and GAT1 Lp values were obtained by measuring Lp in the presence and absence of blockers (phlorizin and SKF89976A). In the presence of the blockers, the Lp values of oocytes expressing SGLT1 and GAT1 were indistinguishable from the Lp of control oocytes. Passive Na+ transport (Na+ leak) was obtained from the blocker-sensitive Na+ currents in the absence of substrates (glucose and GABA). 2. Passive Na+ and water transport through SGLT1 were blocked by phlorizin with the same sensitivity (inhibitory constant (Ki), 3-5 microM). When Na+ was replaced with Li+, phlorizin also inhibited Li+ and water transport, but with a lower affinity (Ki, 100 microM). When Na+ was replaced by choline, which is not transported, the SGLT1 Lp was indistinguishable from that in Na+ or Li+, but in this case water transport was less sensitive to phlorizin. 3. The activation energies (Ea) for passive Na+ and water transport through SGLT1 were 21 and 5 kcal mol-1, respectively. The high Ea for Na+ transport is comparable to that of Na+-glucose cotransport and indicates that the process is dependent on conformational changes of the protein, while the low Ea for water transport is similar to that of water channels (aquaporins). 4. GAT1 also behaved as an SKF89976A-sensitive water channel. We did not observe passive Na+ transport through GAT1. 5. We conclude that passive water and Na+ transport through cotransporters depend on different mechanisms: Na+ transport occurs by a saturable uniport mechanism, and water permeation is through a low conductance water channel. In the case of SGLT1, we suggest that both the water channel and water cotransport could contribute to isotonic fluid transport across the intestinal brush border membrane.

Glutamate-induced suppression of inhibitory synaptic transmission in cultivated hippocampal neurons

In a dissociated culture of rat hippocampal neurons (14 to 24 daysin vitro), modulation effects of glutamate on GABAA-ergic inhibitory transmission were studied with the use of simultaneous patch-clamp whole-cell recording from monosynaptically connected neuron pairs. In all experiments (n=49), 1.5-min-long or longer extracellular application of 0.5 to 100 μM glutamate suppressed evoked inhibitory postsynaptic currents (IPSC). This suppression usually included fast (seconds) and slow (τ=1.3 min) phases. In 83.7% of the cases studied, IPSC did not return to the control values during the entire subsequent recording period (from 10 to 64 min). When glutamate was applied in the presence of blockers of glutamate ionotropic receptors, DL-APV or CNQX, the fast phase of the effect was removed, while some suppression of inhibitory neuronal responses, although weaker, was preserved (n=19); in most cases (73.3%) this residual suppression was slow and long-lasting. It is concluded that both types of glutamate receptors, ionotropic and metabotropic, are involved in modulation of GABAA-ergic synaptic transmission. The first above receptor type provides fast and reversible suppression, while the effect provided by the second type is slow and long-lasting.

Protection From Apoptosis by Steel Factor But Not Interleukin-3 Is Reversed Through Blockade of Calcium Influx

AbstractSteel factor (SLF), the ligand for the c-Kit receptor, protects hemopoietic progenitors and mast cells from apoptosis. We show here that protection of 32D-Kit cells or mast cells from apoptosis by SLF is abrogated through concurrent inhibition of Ca2+influx. In contrast, cell survival promoted by interleukin-3 is not affected by Ca2+ influx blockers. In the presence of blockers, increasing stimulation by SLF leads to greater levels of cell death in the population, indicating that it is the combination of activation by SLF with concurrent blockade of Ca2+ influx that results in apoptosis. The p815 mastocytoma, which expresses a mutated, constitutively active c-kit receptor, dies apoptotically in the presence of Ca2+ influx blockers alone. Ionomycin protects cells from SLF plus blocker-induced apoptosis, confirming specificity for Ca2+ ion blockade in cell death induction. Overexpression of bcl-2, which protects 32D-Kit cells from factor withdrawal, does not protect cells from apoptosis by SLF plus blocker. In contrast, caspase inhibitors YVAD-CHO, DEVD-FMK, and Boc-Asp-FMK protect cells from SLF plus blocker-induced death. These observations highlight the importance of SLF-stimulated Ca2+ influx in the protection of cells from apoptosis and demonstrate a new mechanism for inducing bcl-2 insensitive, caspase-dependent apoptosis through the combination of SLF stimulation with Ca2+ influx blockade.

Protection From Apoptosis by Steel Factor But Not Interleukin-3 Is Reversed Through Blockade of Calcium Influx

Steel factor (SLF), the ligand for the c-Kit receptor, protects hemopoietic progenitors and mast cells from apoptosis. We show here that protection of 32D-Kit cells or mast cells from apoptosis by SLF is abrogated through concurrent inhibition of Ca2+influx. In contrast, cell survival promoted by interleukin-3 is not affected by Ca2+ influx blockers. In the presence of blockers, increasing stimulation by SLF leads to greater levels of cell death in the population, indicating that it is the combination of activation by SLF with concurrent blockade of Ca2+ influx that results in apoptosis. The p815 mastocytoma, which expresses a mutated, constitutively active c-kit receptor, dies apoptotically in the presence of Ca2+ influx blockers alone. Ionomycin protects cells from SLF plus blocker-induced apoptosis, confirming specificity for Ca2+ ion blockade in cell death induction. Overexpression of bcl-2, which protects 32D-Kit cells from factor withdrawal, does not protect cells from apoptosis by SLF plus blocker. In contrast, caspase inhibitors YVAD-CHO, DEVD-FMK, and Boc-Asp-FMK protect cells from SLF plus blocker-induced death. These observations highlight the importance of SLF-stimulated Ca2+ influx in the protection of cells from apoptosis and demonstrate a new mechanism for inducing bcl-2 insensitive, caspase-dependent apoptosis through the combination of SLF stimulation with Ca2+ influx blockade.

Trapping channel block of NMDA-activated responses by amantadine and memantine.

We investigated the mechanisms by which the antiparkinsonian and neuroprotective agents amantadine and memantine inhibit responses to N-methyl-D-aspartic acid (NMDA). Whole cell recordings were performed using cultured rat cortical neurons or Chinese hamster ovary (CHO) cells expressing NMDA receptors. Both amantadine and memantine blocked NMDA-activated channels by binding to a site at which they could be trapped after channel closure and agonist unbinding. For neuronal receptors, the IC50s of amantadine and memantine at -67 mV were 39 and 1.4 microM, respectively. When memantine and agonists were washed off after steady-state block, one-sixth of the blocked channels released rather than trapped the blocker; memantine exhibited "partial trapping." Thus memantine appears to have a lesser tendency to be trapped than do phencyclidine or (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[1,d]cyclihepten-5,1 0-imine (MK-801). We next investigated mechanisms that might underlie partial trapping. Memantine blocked and could be trapped by recombinant NMDA receptors composed of NR1 and either NR2A or NR2B subunits. In these receptors, as in the native receptors, the drug was released from one-sixth of blocked channels rather than being trapped in all of them. The partial trapping we observed therefore was not due to variability in the action of memantine on a heterogeneous population of NMDA receptors in cultured cortical neurons. Amantadine and memantine each noncompetitively inhibited NMDA-activated responses by binding at a second site with roughly 100-fold lower affinity, but this form of inhibition had little effect on the extent to which memantine was trapped. A simple kinetic model of blocker action was used to demonstrate that partial trapping can result if the presence of memantine in the channel affects the gating transitions or agonist affinity of the NMDA receptor. Partial trapping guarantees that during synaptic communication in the presence of blocker, some channels will release the blocker between synaptic responses. The extent to which amantadine and memantine become trapped after channel block thus may influence their therapeutic effects and their modulation of NMDA-receptor-mediated excitatory postsynaptic potentials.

Calcium channels do not play a role in the steroid response to ACTH IN Y1 adrenocortical cells.

Y1 cells derived from mouse tumor zona fasciculata cells (ZF) were used to assess the importance of extracellular (EC) calcium availability via voltage-dependent calcium channels (VDCC) on steroidogenesis. The steroidogenic response to ACTH was investigated in the presence of blockers known to affect both calcium and potassium channels in Y1 cells. Y1 cells respond to either ACTH (100 pM) or cAMP (300 microM) at low EC Ca2+ (1 microM) suggesting that EC Ca2+ is not absolutely necessary for a steroidogenic response. However, increases in Ca2+ from 0.05-2.2 mM induced a small but significant biphasic response, first stimulating then inhibiting steroidogenesis. Nickel and amiloride, blockers of T-type Ca2+ channels in Y1 cells, did not depress ACTH-induced steroidogenesis. The dihydropyridine, nifedipine, which is an L-type channel antagonist did not affect ACTH-induced steroidogenesis while the agonist, Bay K 8644 was consistently inhibitory. Neither pimozide nor omega-conotoxin which suppressed Ca2+ currents in Y1 cells inhibited ACTH-induced steroidogenesis. Depolarization of the membrane which would activate VDCC's was inhibitory rather than stimulatory. The present studies using blockers of both voltage-dependent Ca2+ and K+ channels suggest that EC Ca2+ plays a modulatory role in ACTH-induced steroidogenesis in Y1 cells but the data do not support the concept that activation of voltage dependent calcium channels are an important mechanism for steroidogenesis.

K(+)-channel blockers restore synaptic plasticity in the neuromuscular junction of dunce, a Drosophila learning and memory mutant.

The effects of K(+)-channel blockers on synaptic transmission in dunce (dnc), a Drosophila learning and memory mutant, were investigated. Larvae dnc mutants lack facilitation and post-tetanic potentiation (PTP) at their motor end-plates; dnc mutants are also deficient in a form of phosphodiesterase, and exhibit abnormally high levels of cyclic adenosine 3',5'-monophosphate (cAMP). A two-microelectrode voltage-clamp was used to record end-plate currents and spontaneous end-plate currents from longitudinal ventrolateral third-instar larval muscle. The K(+)-channel blockers 3,4-diaminopyridine (3,4-DAP) and tetraethylammonium (TEA), at micromolar concentrations, caused a reversible decrease in end-plate current amplitudes both in wild-type and mutant end-plates. In the presence of blockers, a period of high-frequency stimulation (tetanus) of the nerve gave way to a transient increase in the end-plate currents of dnc mutants resembling facilitation and PTP in normal end-plates; 3,4-DAP and TEA also restored facilitation and PTP in normal end-plates after incubation with a non-hydrolysable analogue of cAMP (8Br-cAMP). It is suggested that a specific K+ conductance might be relevant to the lack of synaptic plasticity at the dnc neuromuscular synapses.

Block of the cyclic GMP-gated channel of vertebrate rod and cone photoreceptors by l-cis-diltiazem.

Inside-out patches were excised from catfish rod or cone outer segments. Single channel and macroscopic currents were recorded from GMP-gated channels activated by 1 mM cGMP in low divalent buffered saline. Currents were blocked by the application of micromolar concentrations of l-cis-diltiazem to the cytoplasmic side of the patch. The concentration dependence of block indicated that a single molecule was sufficient to block a channel and that all channels were susceptible to block. The dissociation constant for the rod channel was an order of magnitude smaller than for the cone channel, but the voltage dependence of block was nearly identical. The macroscopic current-voltage relation in the presence of blocker was inwardly rectifying and superficially resembled voltage-dependent block by an impermeant blocker occluding the ion-conducting pore of the channel. Block by diltiazem acting from the extracellular side of the channel was investigated by including 5 microM diltiazem in the recording pipette solution. The macroscopic current-voltage relation again showed inward rectification, inconsistent with the idea that diltiazem acts by occluding the pore at the external side. The kinetics of block by diltiazem applied to the intra- and extracellular side were measured in cone patches containing only a single channel. The unbinding rates were similar in both cases, suggesting a single binding site. Differences in the binding rate were consistent with greater accessibility to the binding site from the cytoplasmic side. Block from the cytoplasmic side was independent of pH, suggesting that the state of ionization of diltiazem was not related to its ability to block the channel in a voltage-dependent fashion. These observations are inconsistent with a pore-occluding blocker, but could be explained if the hydrophobic portion of diltiazem partitioned into the hydrophobic core of the channel protein, perhaps altering the gating of the channel.

Effects of L-dopa and L-tyrosine on adrenergic transmission in the canine saphenous vein.

The effects of L-dopa, and L-tyrosine on norepinephrine (NE) and dopamine (DA) content and overflow from adrenergically innervated canine saphenous veins were determined. L-Dopa treatment resulted in large increases in concentration of DA in tissue, and caused significant amounts of DA to overflow into the superfusate both under basal conditions and during electrical stimulation. The DA did not appear to be of vesicular origin, since DA concentrations in superfusate were not altered by removing Ca2+ from the medium. The increased amounts of DA in the junctional cleft did not appear to activate prejunctional dopaminergic or a-adrenergic receptors, since endogenous NE overflow in treated veins was not lower than that observed in untreated veins. Evoked tension responses were also not changed in L-dopa-treated veins indicating that DA did not activate post- synaptic a- or P-adrenergic receptors. Treatment with L-dopa, but not with L-tyrosine, caused small increases in tissue content of NE. Treatment with 5 x 10-5M L-dopa or 5 X 10-4M L-tyrosine attenuated the decrease in NE overflow which was found consistently in untreated veins. However, factors other than lack of precursor must have been responsible for the decrease in NE overflow, since release of NE (measured in the presence of blockers of neuronal and extraneuronal uptakes) did not decrease upon repeated nerve stimulations. L-Dopa is used widely to treat patients with Parkinson's disease (1 -3). Patients only very rarely develop hypertension during L-dopa therapy, although L-dopa is a precursor of L-norepinephrine (L-NE), a potent vasoconstrictor substance. Par- adoxically, hypotension is a rather fre- quent side effect seen during L-dopa treat- ment (1, 2). The hypotensive effects of L- dopa treatment are thought to be mediated by central mechanisms (4), but as pointed out by Westfall (3, effects at the level of the peripheral adrene rgic nerve terminal have not yet been studied and may contrib- ute to the vasodilator effects of L-dopa. In the present experiments we have studied the effects of L-dopa on basal and evoked release of catecholamines at a peripheral neuroeffector junction. The con- centration of L-dopa that was used (10 pg/ml, 5 x M) was one that approxi-

The Influence of Concurrently Administered Theophylline, Ouabain and Hypocapnia on Coronary Flow Perturbations in the Perfused Guinea Pig Heart

Adenosine, potassium ions and hydrogen ions are known to be vasoactive in the coronary circulation. Little is known, however, about the combined effects of these chemicals during reactive hyperemia, flow autoregulation or hypoxic hyperemia. Isolated, perfused guinea pig hearts were used to study the influence of simultaneously administered theophylline, ouabain and alkalosis on coronary flow responses (occlusive hyperemia, autoregulation, hypoxic hyperemia) thought to be contributed to by the above chemicals. Retrograde aortic inflow was monitored electromagnetically in the absence and presence of concurrently administered theophylline (5 x 10(-5) M), ouabain (1.4 x 10(-7) M), and alkalosis (perfusate pH 7.69). Upon release of a 30-sec inflow occlusion (in the presence of the above agents) mean peak coronary flow was modestly, but significantly, reduced. Volume flow rate and time for flow to return 50% towards control were reduced in the presence of the above agents, but failed to return to pre-experimental values upon removal of blockers. When perfusion pressure was increased from 65 to 95 cm H2O (antagonists present), coronary myogenic autoregulation was significantly enhanced. Upon decreasing pressure from 95 to 35 cm H2O, in the presence of blockers, calculated coronary resistance fell from 13 +/- 0.6 to 9.5 +/- 0.5 cm H2O/ml/min. The latter value was significantly higher than that seen in the absence of test agents. No difference in the maximum mean coronary flow achieved by hypoxia (20% O2-5% Co2-balance N2) was seen in the absence or presence of blockers. In conclusion, these experiments provide some indirect support for the involvement of the above metabolites in reactive hyperemia and as antagonists of myogenic blood flow autoregulation. Little evidence for their involvement in hypoxic hyperemia is available from this study.