A non-capacitative pathway activated by arachidonic acid is the major Ca2+ entry mechanism in rat A7r5 smooth muscle cells stimulated with low concentrations of vasopressin.
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1. Depletion of the Ca2+ stores of A7r5 cells stimulated Ca2+, though not Sr2+, entry. Vasopressin (AVP) or platelet-derived growth factor (PDGF) stimulated Sr2+ entry. The cells therefore express a capacitative pathway activated by empty stores and a non-capacitative pathway stimulated by receptors; only the former is permeable to Mn2+ and only the latter to Sr2+. 2. Neither empty stores nor inositol 1,4,5-trisphosphate (InsP3) binding to its receptors are required for activation of the non-capacitative pathway, because microinjection of cells with heparin prevented PDGF-evoked Ca2+ mobilization but not Sr2+ entry. 3. Low concentrations of Gd3+ irreversibly blocked capacitative Ca2+ entry without affecting AVP-evoked Sr2+ entry. After inhibition of the capacitative pathway with Gd3+, AVP evoked a substantial increase in cytosolic [Ca2+], confirming that the non-capacitative pathway can evoke a significant increase in cytosolic [Ca2+]. 4. Arachidonic acid mimicked the effect of AVP on Sr2+ entry without stimulating Mn2+ entry; the Sr2+ entry was inhibited by 100 microM Gd3+, but not by 1 microM Gd3+ which completely inhibited capacitative Ca2+ entry. The effects of arachidonic acid did not require its metabolism. 5. AVP-evoked Sr2+ entry was unaffected by isotetrandrine, an inhibitor of G protein-coupled phospholipase A2. U73122, an inhibitor of phosphoinositidase C, inhibited AVP-evoked formation of inositol phosphates and Sr2+ entry. The effects of phorbol esters and Ro31-8220 (a protein kinase C inhibitor) established that protein kinase C did not mediate the effects of AVP on the non-capacitative pathway. An inhibitor of diacylglycerol lipase, RHC-80267, inhibited AVP-evoked Sr2+ entry without affecting capacitative Ca2+ entry or release of Ca2+ stores. 6. Selective inhibition of capacitative Ca2+ entry with Gd3+ revealed that the non-capacitative pathway is the major route for the Ca2+ entry evoked by low AVP concentrations. 7. We conclude that in A7r5 cells, the Ca2+ entry evoked by low concentrations of AVP is mediated largely by a non-capacitative pathway directly regulated by arachidonic acid produced by the sequential activities of phosphoinositidase C and diacylglycerol lipase. (+info)
Stimulation of Na+-alanine cotransport activates a voltage-dependent conductance in single proximal tubule cells isolated from frog kidney.
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1. The swelling induced by Na+-alanine cotransport in proximal tubule cells of the frog kidney is followed by regulatory volume decrease (RVD). This RVD is inhibited by gadolinium (Gd3+), an inhibitor of stretch-activated channels, but is independent of extracellular Ca2+. 2. In this study, the whole cell patch clamp technique was utilized to examine the effect of Na+-alanine cotransport on two previously identified volume- and Gd3+-sensitive conductances. One conductance is voltage dependent and anion selective (GVD) whilst the other is voltage independent and cation selective (GVI). 3. Addition of 5 mM L-alanine to the bathing solution increased the whole cell conductance and gave a positive (depolarizing) shift in the reversal potential (Vrev, equivalent to the membrane potential in current-clamped cells) consistent with activation of Na+-alanine cotransport. Vrev shifted from -36 +/- 4.9 to +12.9 +/- 4.2 mV (n = 15). 4. In the presence of alanine, the total whole cell conductance had several components including the cotransporter conductance and GVD and GVI. These conductances were separated using Gd3+, which inhibits both GVD and GVI, and the time dependency of GVD. Of these two volume-sensitive conductances, L-alanine elicited a specific increase in GVD, whereas GVI was unaffected. 5. The L-alanine-induced activation of GVD was significantly reduced when cells were incubated in a hypertonic bathing solution. 6. In summary, in single proximal tubule cells isolated from frog kidney, on stimulation of Na+-alanine cotransport GVD is activated, while GVI is unaffected. Taken with other evidence, this suggests that GVD is activated by cell swelling, consequent upon alanine entry, and may play a role as an anion efflux pathway during alanine-induced volume regulation. (+info)
Aluminum is a weak agonist for the calcium-sensing receptor.
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BACKGROUND: Aluminum (Al3+) has diverse biological effects mediated through activation of a putative extracellular cation-sensing receptor. A recently identified calcium-sensing receptor (CaSR), which has been identified in target tissues for Al3+, may transduce some of the biological effects of Al3+. METHODS: To test this possibility, we transfected human embryonic kidney 293 (HEK 293) cells with a cDNA encoding the rat CaSR and evaluated CaSR expression by Western blot analysis and function by measurement of intracellular calcium ([Ca2+]i) levels and inositol monophosphate (IP1) generation following stimulation with Al3+ and a panel of CaSR agonists. RESULTS: The CaSR protein was detected by immunoblot analysis in cells transfected with the CaSR cDNA but not in nontransfected HEK 293 cells. In addition, [Ca2+]i levels and IP1 generation were enhanced in a dose-dependent fashion by additions of the CaSR agonists calcium (Ca2+), magnesium (Mg2+), gadolinium (Gd3+), and neomycin only in cells transfected with CaSR. To determine if Al3+ activated CaSR, we stimulated cells transfected with rat CaSR with 10 microM to 1 mM concentrations of Al3+. Concentrations of Al3+ in the range of 10 microM to 100 microM had no effect on [Ca2+]i levels or IP1 generation. In contrast, 1 mM Al3+ induced small but significant increases in both parameters. Whereas Gd3+ antagonized calcium-mediated activation of CaSR, pretreatment with Al3+ failed to block subsequent activation of rat CaSR by Ca2+, suggesting a distinct mechanism of Al3+ action. CONCLUSION: Al3+ is not a potent agonist for CaSR. Because Al3+ affects a variety of target tissues at micromolar concentrations, it seems unlikely that CaSR mediates these cellular actions of Al3+. (+info)
Signal transduction in spontaneous myogenic tone in isolated arterioles from rat skeletal muscle.
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OBJECTIVE: The mechanism of spontaneous myogenic tone was investigated in isolated arteriolar segments. METHODS: Arterioles were isolated from rat cremaster muscle. Segments were endothelium-denuded and mounted in a pressure myograph at 75 mmHg. Under this condition, segments spontaneously constricted from a passive diameter of 167 +/- 3 to 82 +/- 4 microns (n = 41). The effects of several inhibitors were tested on the maintenance of myogenic tone. RESULTS: Gadolinium (10(-6)-10(-4) M), a putative inhibitor of stretch-activated cation channels, was ineffective. The phospholipase C (PLC) inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC) induced a dose-dependent inhibition of tone. NCDC inhibited phenylephrine- (10(-6) M), but not potassium buffer-induced (100 mM) constriction. The protein kinase C (PKC) inhibitors staurosporine, chelerythrine and calphostin C inhibited myogenic tone in a concentration-dependent manner. At an intermediate concentration, calphostin C selectively inhibited phenylephrine-induced constriction. However, all PKC inhibitors abolished responses to phenylephrine and potassium buffer at higher concentrations. The cytochrome P450 inhibitor 17-ODYA (0.3-3 x 10(-6) M) did not inhibit myogenic tone. CONCLUSIONS: No evidence was found for a role of gadolinium-sensitive, stretch-activated cation channels or cytochrome P450 metabolites. On the other hand, both PLC and PKC contribute to the maintenance of myogenic tone. (+info)
Contractile activity in intestinal muscle evokes action potential discharge in guinea-pig myenteric neurons.
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1. The process by which stretch of the external muscle of the intestine leads to excitation of myenteric neurons was investigated by intracellular recording from neurons in isolated longitudinal muscle-myenteric plexus preparations from the guinea-pig. 2. Intestinal muscle that was stretched by 40 % beyond its resting size in either the longitudinal or circular direction contracted irregularly. Both multipolar, Dogiel type II, neurons and uniaxonal neurons generated action potentials in stretched tissue. Action potentials persisted when the membrane potential was hyperpolarized by passing current through the recording electrode for 10 of 14 Dogiel type II neurons and 1 of 18 uniaxonal neurons, indicating that the action potentials originated in the processes of these neurons. For the remaining four Dogiel type II and 17 uniaxonal neurons, the action potentials were abolished, suggesting that they were the result of synaptic activation of the cell bodies. 3. Neurons did not fire action potentials when the muscle was paralysed by nicardipine (3 microM), even when the preparations were simultaneously stretched by 50 % beyond resting length in longitudinal and circular directions. Spontaneous action potentials were not recorded in unstretched (slack) tissue, but when the L-type calcium channel agonist (-)-Bay K 8644 (1 microM) was added, the muscle contracted and action potentials were observed in Dogiel type II neurons and uniaxonal neurons. 4. The proteolytic enzyme dispase (1 mg ml-1) added to preparations that were stretched 40 % beyond slack width caused the myenteric plexus to lift away from the muscle, but did not prevent muscle contraction. In the presence of dispase, the neurons ceased firing action potentials spontaneously, although action potentials could still be evoked by intracellular current pulses. After the action of dispase, (-)-Bay K 8644 (1 microM) contracted the muscle but did not cause neurons to fire action potentials. 5. Gadolinium ions (1 microM), which block some stretch activated ion channels, stopped muscle contraction and prevented action potential firing in tissue stretched by 40 %. However, when (-)-Bay K 8644 (1 microM) was added in the presence of gadolinium, the muscle again contracted and action potentials were recorded from myenteric neurons. 6. Stretching the tissue 40 % beyond its slack width caused action potential firing in preparations that had been extrinsically denervated and in which time had been allowed for the cut axons to degenerate. 7. The present results lead to the following hypotheses. The neural response to stretching depends on the opening of stretch activated channels in the muscle, muscle contraction in response to this opening, and mechanical communication from the contracting muscle to myenteric neurons. Distortion of sensitive sites in the processes of the neurons opens channels to initiate action potentials that are propagated to the soma, where they are recorded. Neurons are also excited indirectly by slow synaptic transmission from neurons that respond directly to distortion. (+info)
Stretch-activated single K+ channels account for whole-cell currents elicited by swelling.
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Functionally significant stretch-activated ion channels have been clearly identified in excitable cells. Although single-channel studies suggest their expression in other cell types, their activity in the whole-cell configuration has not been shown. This discrepancy makes their physiological significance doubtful and suggests that their mechanical activation is artifactual. Possible roles for these molecules in nonexcitable cells are acute cell-volume regulation and, in epithelial cells, the complex adjustment of ion fluxes across individual cell membranes when the rate of transepithelial transport changes. We report the results of experiments on isolated epithelial cells expressing in the basolateral membrane stretch-activated K+ channels demonstrable by the cell-attached patch-clamp technique. In these cells, reversible whole-cell currents were elicited by both isosmotic and hyposmotic cell swelling. Cation selectivity and block by inorganic agents were the same for single-channel and whole-cell currents, indicating that the same entity underlies single-channel and whole-cell currents and that the single-channel events are not artifactual. In these cells, when the rate of apical-membrane NaCl entry increases, the cell Na+ content and volume also increase, stimulating the Na+,K+-ATPase at the basolateral membrane, i.e., both Na+ extrusion and K+ uptake increase. We speculate that, under these conditions, the parallel activation of basolateral K+ channels (by the swelling) elevates conductive K+ loss, tending to maintain the cell K+ content constant ("pump-leak parallelism"). This study describes a physiologically relevant stretch-activated channel, at both the single-channel and whole-cell levels, in a nonneural cell type. (+info)
Spatial mapping of T2 and gadolinium-enhancing T1 lesion volumes in multiple sclerosis: evidence for distinct mechanisms of lesion genesis?
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It is generally believed that most T2-weighted (T2) lesions in the central white matter of patients with multiple sclerosis begin with a variable period of T1-weighted (T1) gadolinium (Gd) enhancement and that T1 Gd-enhancing and T2 lesions represent stages of a single pathological process. Lesion probability maps can be used to test this hypothesis by providing a quantitative description of the spatial distribution of these two types of lesions across a patient population. The simplest prediction of this hypothesis would be that the spatial distributions of T1 Gd-enhancing and T2 lesions are identical. We generated T1 Gd-enhancing and T2 lesion probability maps from 19 patients with relapsing-remitting multiple sclerosis. There was a significantly higher probability (P = 0.001) for T2 lesions to be found in the central relative to the peripheral white matter (risk ratio 4.5), although the relative distribution of T1 Gd-enhancing lesions was not significantly different (P = 0.7) between central and peripheral white matter regions (risk ratio 0.6). Longitudinal data on the same population were used to demonstrate a similar distribution asymmetry between new T1 Gd-enhancing and new T2 lesions that developed over the course of 1 year. Alternative hypotheses to explain this observation were tested. We found no spatial difference in the likelihood of development of persistent T2 lesions following T1 Gd enhancement. The relative distribution of T1 Gd-enhancing lesions was shown to be independent of the dose of Gd contrast agent and the frequency of scanning. Our findings suggest that a proportion of the periventricular T2 lesion volume may arise from mechanisms other than those associated with early breakdown of the blood-brain barrier leading to T1 Gd enhancement. (+info)
Molecular and functional identification of a Ca2+ (polyvalent cation)-sensing receptor in rat pancreas.
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The balance between the concentrations of free ionized Ca2+ and bicarbonate in pancreatic juice is of critical importance in preventing the formation of calcium carbonate stones. How the pancreas regulates the ionic composition and the level of Ca2+ saturation in an alkaline environment such as the pancreatic juice is not known. Because of the tight cause-effect relationship between Ca2+ concentration and lithogenicity, and because hypercalcemia is proposed as an etiologic factor for several pancreatic diseases, we have investigated whether pancreatic tissues express a Ca2+-sensing receptor (CaR) similar to that recently identified in parathyroid tissue. Using reverse transcriptase-polymerase chain reaction and immunofluorescence microscopy, we demonstrate the presence of a CaR-like molecule in rat pancreatic acinar cells, pancreatic ducts, and islets of Langerhans. Functional studies, in which intracellular free Ca2+ concentration was measured in isolated acinar cells and interlobular ducts, show that both cell types are responsive to the CaR agonist gadolinium (Gd3+) and to changes in extracellular Ca2+ concentration. We also assessed the effects of CaR stimulation on physiological HCO3- secretion from ducts by making measurements of intracellular pH. Luminal Gd3+ is a potent stimulus for HCO3- secretion, being equally as effective as raising intracellular cAMP with forskolin. These results suggest that the CaR in the exocrine pancreas monitors the Ca2+ concentration in the pancreatic juice, and might therefore be involved in regulating the level of Ca2+ in the lumen, both under basal conditions and during hormonal stimulation. The failure of this mechanism might lead to pancreatic stone formation and even to pancreatitis. (+info)