Cellular Calcium Concentration Research Articles

A subpopulation of mitochondria prevents cytosolic calcium overload in endothelial cells after cold ischemia/reperfusion.

Calcium represents a key mediator of cold ischemia/reperfusion (CIR) injury presumably by affecting mitochondrial function. In this study, we investigated cellular and mitochondrial changes of calcium homeostasis in sublethally damaged human endothelial cells. Changes in cellular and mitochondrial calcium concentrations were studied after cold ischemia in University of Wisconsin solution for 12 hr and reperfusion in ringer solution. Cytosolic-free calcium concentration ([Ca2+]c) and mitochondrial-free calcium content ([Ca2+]m) were analyzed by fura-2 and rhod-2 fluorescence, respectively. Pretreatment of cells with ruthenium red (RR) or a H+-ionophore was used to inhibit mitochondrial calcium uptake. Mitochondrial membrane potential (DeltaPsim) was measured by 5,5',6,6'-tetrachloro- 1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide and 3,3'-dihexyloxacarbocyanine iodide fluorescence. Twelve-hr cold ischemia did not induce apoptosis in endothelial cells. In such sublethally damaged cells, [Ca2+]c rose from approximately 20 nmol/L after cold ischemia to approximately 120 nmol/L during reperfusion. Pretreatment with RR leads to an approximately 5-fold rise in [Ca2+]c. Image analysis revealed a significant increase of [Ca2+]m in a subpopulation of mitochondria during reperfusion. This was not the case in RR-pretreated cells. DeltaPsim decreased significantly during cold ischemia and was sustained during reperfusion. The loss of DeltaPsim can be related to a reduced portion of mitochondria exhibiting high DeltaPsim. Our results suggest that cytosolic calcium influx during CIR is buffered by a selective portion of mitochondria in human umbilical vein endothelial cells. These mitochondria protect cells against cytosolic calcium overload and probably against subsequent cell injury.

Effects of ethoxyquin on the blood composition of turbot, Scophthalmus maximus L.

Ethoxyquin (6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline (EQ) is a synthetic antioxidant used for preventing rancidity in animal foodstuffs. Three groups of ten fish were given a diet containing respectively 75 (control group with the commercial food), 200 and 400 ppm EQ for 16 days. The control group had a plasma osmolality and chloride concentration within the normal range of marine teleosts, but sodium concentrations of only about 110 mM, indicating the presence in the plasma of substantial amounts of another cation. Fish given food with 400 ppm EQ displayed a 70 mM increase in the plasma concentration of sodium. This indicates that EQ has disturbed the iono-regulatory mechanisms, probably by reducing the ATP production or inhibiting directly the Na/K-ATPase in the gills. The large increase in plasma sodium concentration was not accompanied by any significant increase in plasma osmolality, indicating that at least a part of the sodium added to the plasma is made osmotically inactive. In spite of the elevated plasma sodium concentration, the sodium content of erythrocytes of the 400-ppm EQ fish was reduced to half, while the content of calcium was unaffected. The transmembrane energy gradient of sodium in the EQ exposed turbot obviously increased, allowing them to use a sodium coupled antiport system to keep the cellular calcium content low when the Ca-ATPases is blocked. A mechanism of this kind is also likely to be important to turbot that experience hypoxia under natural conditions. The 400-ppm group also displayed a substantial increase in liver weight, but the physiological significance of this effect is not clear. The leucocyte counts indicated the absence of obvious immunological effects.

Membrane, intracellular, and plasma magnesium and calcium concentrations in preeclampsia

Changes in intracellular calcium and magnesium concentrations seem to be involved in the pathogenesis of preeclampsia, whereas the role of cell membranes has not been studied in detail yet. To investigate the changes in calcium and magnesium metabolism in normal pregnancy and preeclampsia, plasma, intracellular, and membrane calcium and magnesium concentrations were determined in a clinical study. Twenty-five control, 18 untreated healthy pregnant, and 16 nulliparas preeclamptic women were investigated. Plasma, cellular, and membrane (erythrocytes) calcium and magnesium contents were measured by atomic absorption spectroscopy. Plasma and intracellular magnesium concentrations were significantly lower in the healthy pregnant group and the preeclamptic group as compared to controls ( P < .01). In erythrocyte membranes magnesium content was found significantly decreased in the preeclamptic women as compared to healthy subjects ( P < .001). There was a significant decrease in the plasma calcium concentration in the preeclamptic group compared to controls or healthy pregnant women ( P < .05). Membranous calcium content was significantly increased in the preeclamptic group versus controls or healthy pregnant women ( P < .001) and an inverse correlation with membranous magnesium content was found (r = −0.79, P < .01). Lowered plasma, intracellular, and membrane magnesium concentrations in preeclampsia may contribute to the development in hypertension in pregnancy. In addition, a disturbed calcium homeostasis is observed in preeclampsia.

Thiopental attenuates hypoxic changes of electrophysiology, biochemistry, and morphology in rat hippocampal slice CA1 pyramidal cells.

Thiopental has been shown to protect against cerebral ischemic damage; however, it has undesirable side effects. We have examined how thiopental alters histological, physiological, and biochemical changes during and after hypoxia. These experiments should enable the discovery of agents that share some of the beneficial effects of thiopental. We made intracellular recordings and measured ATP, sodium, potassium, and calcium concentrations from CA1 pyramidal cells in rat hippocampal slices subjected to 10 minutes of hypoxia with and without 600 micromol/L thiopental. Thiopental delayed the time until complete depolarization (21+/-3 versus 11+/-2 minutes for treated versus untreated slices, respectively) and attenuated the level of depolarization at 10 minutes of hypoxia (-33+/-6 versus -12+/-5 mV). There was improved recovery of the resting potential after 10 minutes of hypoxia in slices treated with thiopental (89% versus 31% recovery). Thiopental attenuated the changes in sodium (140% versus 193% of prehypoxic concentration), potassium (62% versus 46%), and calcium (111% versus 197%) during 10 minutes of hypoxia. There was only a small effect on ATP (18% versus 8%). The percentage of cells showing clear histological damage was decreased by thiopental (45% versus 71%), and thiopental improved protein synthesis after hypoxia (75% versus 20%). Thiopental attenuates neuronal depolarization, an increase in cellular sodium and calcium concentrations, and a decrease in cellular potassium and ATP concentrations during hypoxia. These effects may explain the reduced histological, protein synthetic, and electrophysiological damage to CA1 pyramidal cells after hypoxia with thiopental.

Calcium accentuates injury induced by ethanol in human gastric cells

The mechanism(s) whereby ethanol induces cellular injury remains poorly understood. Furthermore, the role of calcium in gastric mucosal injury under in vitro conditions is poorly defined. The major objectives of this study were to (1) define the temporal relationship between intracellular calcium accumulation induced by ethanol and cellular injury, (2) characterize the mechanism(s) whereby ethanol increases cellular calcium content, and (3) determine whether calcium removal would attenuate ethanol-induced cellular injury. Human gastric cells (AGS) were used for all experiments. Sustained intracellular calcium accumulation induced by ethanol, but not transient changes, preceded and directly correlated with cellular injury. Cells exposed to damaging concentrations of ethanol demonstrated an initial calcium surge that appeared to be a consequence of inositol 1,4,5-triphosphate (IP 3) generation and subsequent internal store release followed by a sustained plateau resulting from extracellular calcium influx through store-operated calcium channels. Finally, both morphologic (cellular injury) and functional (clearance of bovine serum albumin) changes induced by ethanol were significantly attenuated when extracellular Ca ++ influx was prevented, and further decreased when intracellular Ca ++ stores were depleted. These data indicate that calcium plays a significant role in cellular injury induced by ethanol.

Characterization of positive inotropic effect of colforsin dapropate hydrochloride, a water-soluble forskolin derivative, in isolated adult rat cardiomyocytes

The present study was undertaken to characterize the positive inotropic action of colforsin daropate [corrected] hydrochloride (NKH477), a novel water-soluble forskolin derivative, on isolated cardiomyocytes of adult rats. Simultaneous measurements of cellular contraction and intracellular calcium concentration ([Ca2+]i) were carried out. The effects of isoprenaline and ouabain on these parameters were also determined for comparison. The contraction and maximum [Ca2+]i of NKH477-, isoprenaline-, or ouabain-treated cells were increased concentration dependently. Peak shortening of NKH477-treated cells was positively correlated with the shortening velocity and inversely with the time to peak shortening. Maximum, but not minimum, [Ca2+]i in NKH477-treated cells was correlated with the rate of increase in [Ca2+]i and inversely with the time to maximum [Ca2+]i. Similar results were obtained with isoprenaline. In contrast, ouabain increased both maximum and minimum [Ca2+]i. Treatment with either NKH477 or isoprenaline increased cellular cAMP content, but treatment with ouabain did not. These results suggest that the positive inotropic action of NKH477 is associated with an increase in [Ca2+]i and acceleration of its kinetics.

Alteration in cellular calcium and mitochondrial functions in the rat liver during cold preservation.

Preservation injury is multifactorial and its mechanism is still incompletely defined. Calcium may play an important role in preservation injury. The effects of hypothermia on cytosolic free calcium concentration ([Ca2+]I) and total cellular calcium content in isolated rat hepatocytes were investigated by using fura-2 fluorescence and atomic absorption spectroscopy. Fura-2 loaded cells were placed into a prechilled (7 degrees C) cuvette equipped with a stirrer or preserved in the University of Wisconsin (UW) solution for up to 48 hr. In some experiments, cells were pretreated with inhibitors of Ca2+ release from mitochondria (m-iodobenzylguanidine [MIBG]) and from endoplasmic reticulum (ryanodine [RYA]) for 20 min at 37 degrees C. Mitochondrial functions after preservation were evaluated by measuring ATP and respiratory rates. Cooling to 7 degrees C caused a rapid increase in [Ca2+]I that was substantially blocked by MIBG and RYA pretreatment. The elevated calcium gradually leaked out of the cells into the Ca2+-free medium. In long-term storage of the cells in the UW solution, there was a marked decrease in both cytosolic free calcium and total cellular calcium. Pretreatment of the livers with MIBG before cold preservation in the UW solution resulted in a stimulation of ATP regeneration in tissue slices. MIBG pretreatment also improved mitochondrial respiratory functions after cold preservation. Thus, the loss of mitochondrial function after liver preservation in the UW solution may be related to the effects of hypothermia on calcium metabolism. Approaches to help maintain calcium homeostasis during storage may improve organ preservation.

Altered monocyte calcium dynamics in sepsis.

This study was designed to evaluate monocyte calcium concentration and mobilization in normal and septic surgical patients. Monocytes were isolated from 15 "septic" surgical patients, washed, and loaded with the fluorescent calcium chelator, FURA-2. Monocytes from 20 normal volunteers served as controls. Intracellular calcium concentration ([Ca2+]i) was measured by means of fluorescent spectrophotometry before, during, and after stimulation with concanavalin A. Differences were evaluated for statistical significance by analysis of variance. The study was repeated using normal monocytes preincubated in "septic" serum and "septic" monocytes preincubated in normal serum. Additional paired whole blood specimens were obtained from the control group and were incubated with Escherichia coli endotoxin. Monocytes were then isolated and evaluated as described. Sepsis was associated with significantly low resting monocyte calcium concentrations. Although concanavalin A stimulation resulted in marked calcium mobilization in both normal and septic cells, final cellular calcium concentration was significantly lower in the stimulated "septic" monocytes. Similar alterations were seen in normal cells incubated with septic serum, but could not be reproduced by incubation with endotoxin. This deficiency could not be corrected in septic cells incubated in normal serum. Sepsis is associated with a significant alteration of monocyte calcium dynamics in both resting and stimulated cells. These changes appear to be modulated by a serum factor other than endotoxin.

Calcium uptake in mosses and its role in stone biodeterioration

The biodeterioration of stone substrata by bryophytes (Musci and Hepaticae) is due to biogeochemical and biogeophysical mechanisms. In this work the biogeochemical damage caused by epilithic moss species, found in archaeological sites of Rome, was investigated. The cellular calcium content in specimens of Grimmia pulvinata (Hedw.) Sm. was analysed using a sequential elution technique. The analyses were carried out on moss specimens sampled from different lithotypes and in different seasons in order to measure the calcium concentration both in relation to the mineral composition of the stone and to the plant physiology. The highest calcium concentration of the extracellular exchangeable fraction was detected in the samples grown on marble and in the waterlogged specimen sampled in spring time.

Biological assays for C-X-C chemokines

Publisher Summary Chemokines play a major role in mobilizing leukocytes to ward off attacks by invading pathogens. Each of the two major classes of chemokines is selective for a particular group of immune cells. This chapter discusses the assays available to measure the biological activities of one of these groups of chemoattractants, the C-X-C chemokines. The C-X-C chemokines that include interleukin-8 (IL-8) and melanoma growth stimulating activity (MGSA) preferentially attract neutrophils and induce their activation by producing changes in neutrophil shape, transient increases in cellular calcium concentration, and the upregulation of surface adhesion proteins. These chemokines play a major role in acute inflammatory responses. The chapter describes a procedure for isolating neutrophils from human blood and then discusses the biological effect of C-X-C chemokines on these cells as measured in a number of assays, including upregulation of CD18 adhesion proteins, elastase and β-glucuronidase release assays, and the oxidative burst.

Extracellular ATP promotes cellular growth of renal inner medullary collecting duct cells mediated via P2u receptors.

The present study was undertaken to determine whether extracellular adenosine 5'-triphosphate (ATP) promotes cellular proliferation of cultured rat renal inner medullary collecting duct cells. Extracellular ATP increased inositol 1,4,5-triphosphate (IP3) production and cellular free calcium concentration - [Ca2+]i - in a dose-dependent manner. ATP also caused a transient cellular acidification. Extracellular ATP activated mitogen-activated protein (MAP) kinase and [3H]thymidine incorporation in a dose-dependent manner. However, such effects were not obtained with adenosine 5'-diphosphate, adenosine monophosphate, and adenosine. In addition, uridine triphosphate, a P(2u) purinergic agonist, increased IP3 production and activated MAP kinase. 2-Methylthio ATP, a P(2y) purinergic agonist, also increased IP3 production, but did not affect the MAP kinase activity. We also examined the effect of arginine vasopressin on cellular growth. Arginine vasopressin did not alter MAP kinase activity and [3H]thymidine incorporation in cultured rat renal inner medullary collecting duct cells. These results indicate that extracellular ATP activates phospholipase C mediated through P(2u) and P(2y) purinergic receptors and promotes cellular proliferation mediated through P(2u) purinergic receptors in renal inner medullary collecting duct cells.

Change in Expression of Heart Carnitine Palmitoyltransferase I Isoforms with Electrical Stimulation of Cultured Rat Neonatal Cardiac Myocytes

Electrical stimulation of neonatal rat cardiac myocytes in culture produces increases in myocyte size (hypertrophy) and organization of actin into myofibrillar arrays. The maturation of the cells is associated with enhanced contractile parameters and cellular calcium content. The numbers and intensity of cellular mitochondrial profiles increase, as measured by scanning laser confocal microscopy. Consistent with the hypertrophic response is increased cellular content of beta-myosin heavy chain and cytochrome oxidase subunit Va messages, as well as increases in cytochrome oxidase activity in the stimulated cardiac myocytes. Myocyte contractile capacity is associated with increased expression of the muscle carnitine palmitoyltransferase (CPT-I) isoform as measured by Northern analysis, immunoblotting, and altered sensitivity of CPT-I activity to malonyl-CoA in the stimulated cells. The data suggest that a switch from the liver isoform of CPT-I, prominent in the neonatal rat heart, to the muscle CPT-I which predominates in adult rat heart, takes place in the neonatal cardiac myocytes over the same time period as the hypertrophic-mediated changes in myofibrillar assembly and increased contractile activity.

Effects of in vivo vanadate administration on calcium exchange and contractile force of rat ventricular myocardium

The aim of this study was to evaluate the effect of a 14-day intragastric administration of Na 3VO 4 (0.03 mmol/kg daily) on calcium metabolism and contractile force of rat myocardium. Left ventricular pressure as well as its first derivative ( dP dt ) were registered with a balloon inserted to this ventricle and the cellular content of exchangeable calcium with the aid of 45Ca 2+. Left ventricular pressure of the hearts exposed to vanadium was 11.8 ± 0.2 kPa and was lower by 23% in comparison with control hearts (without vanadate), and its first derivative was 131.01 ± 2.8 kPa/s (lower by 36.4%). The cellular content of exchangeable calcium in myocardium of rats treated with vanadium was 1.305 ± 0.173 and 2.019 +- 0.231 mmol/kg of wet weight (w.w.) in the stimulated and in the rested (for the last 10 min) ventricles, respectively. Accordingly, in the control group, the Ca content in stimulated hearts was lower by 0.384 mmol/kg w.w., and in the rested ones it was lower by 0.715. This indicates that the myocardial contractile force decreases, in spite of the fact that the content of calcium is considerably higher than that in the control group. These results also show a toxic effect of vanadate on the myocardium, probably due to large intracellular accumulation of calcium and cell damage.

Swelling of the vesicle is prerequisite for PTH secretion.

Unlike most secretory cells, high extra cellular calcium inhibits rather than stimulates hormonal secretion in several cells such as parathyroid cells, Juxtaglomerular cells and osteoclast. To gain further insight into the common but unique stimulus-secretion coupling mechanism in these cells, bovine parathyroid slices were incubated in various conditions of Krebs-Ringer (KR) solution containing essential amino acids. Parathyroid cells showed the inverse dependency of secretion on extra cellular calcium concentration as we expected. Ammonium acetate overcame the inhibitory effect of 2.5 mM of calcium and the maximum effect was as much as the five times of the basal value, while there was a little additive effect under 0 mM CaCl2. PTH secretion was biphasic according to the change of extra cellular osmolarity and the lowest response was observed at 300 mOsm/l. In Na-rich KR solution, high concentration of nigericin (> 10(-4)M) completely overcame the inhibitory effect of 2.5 mM CaCl2 and the maximum stimulatory effect was 8 times greater whereas it was only 2 times greater without CaCl2. In K-rich KR solution that abolished the K-gradient between the extra cellular solution and the cytoplasm, the rate of PTH secretion increased, and furthermore the addition of nigericin increased the rate of secretion significantly. The results above suggested that the osmotic swelling of the secretory vesicle in parathyroid cells might promote exocytosis as in Juxtaglomerular cells. We propose that the swelling of the vesicle is also prerequisite for secretion in several cells inhibited paradoxically by Ca++, whatever the signal transduction pathway for swelling of the secretory granules induced by the lowering of Ca++ in cytoplasm are.

Biochemical basis of hepatocellular injury.

The hepatotoxic response elicited by a chemical agent depends on the concentration of the toxicant (parent compound or metabolite) delivered to the hepatocytes across the liver acinus via blood flow. Hepatotoxicants produce characteristic patterns of cytolethality in specific zones of the acinus due to the differential expression of enzymes and the concentration gradients of cofactors and toxicant in blood across the acinus. Most hepatotoxic chemicals produce necrosis, characterized by swelling in contiguous tracts of cells and inflammation. This process has been contrasted with apoptosis, where cells and organelles condense in an orderly manner under genetic control. Biotransformation can activate a chemical to a toxic metabolite or decrease toxicity. Quantitative or qualitative species differences in biotransformation pathways can lead to significant species differences in hepatotoxicity. Fasted rodents are more susceptible to the hepatotoxic effects of many chemicals due to glutathione depletion and cytochrome P-450 induction. Freshly isolated hepatocytes are the most widely used in vitro system to study mechanisms of cell death. Hepatotoxicants can interact directly with cell macromolecules or via a reactive metabolite. The reactive metabolite can alkylate critical cellular macromolecules or induce oxidative stress. These interactions generally lead to a loss of calcium homeostasis prior to plasma membrane lysis. Mitochondria have been shown to be important cellular targets for many hepatotoxicants. Decreasing hepatocellular adenosine triphosphate concentrations compromise the plasma membrane calcium pump, leading to increased cellular calcium concentrations. Calcium-dependent endonucleases produce double-strand breaks in DNA before cell lysis. These biochemical pathways induced by necrosis-causing toxicants are similar to the biochemical pathways involved in apoptosis, suggesting that apoptosis and necrosis differ in intracellular and extracellular control points rather than in the biochemistry involved in cell death.

Defence Mechanisms Protecting Glial Cell Cultures against the Toxicity of Lead

— Several defence mechanisms against the toxicity of triethyllead (TEL) in cultures of glial cells have been studied. In contrast to cultures of glioma C6 cells, primary cultures of glial cells generated a resistance to the toxicity of TEL, which increased with the age of the cultures. When the time of exposure of glial cells was extended from 24 hours to 6 days and the cells were exposed to increasing doses of TEL, the activities of several metabolic enzymes were stimulated by concentrations of TEL over 10-7M. Among these were glucose-6-phosphate-dehydrogenase and acid phosphatase. On the other hand, lactate dehydrogenase activity was not significantly affected under these experimental conditions. When the cultures were exposed to 10 8M TEL, the cellular calcium content increased and the potassium content fell. This indicates that the activation of metabolic enzymes is a response to early events in the toxic process, which include disturbances in calcium metabolism. When glial cells were incubated with lead and selenium simultaneously, the uptake of lead into the cells was reduced, and the cellular calcium content was increased by selenium.

Time-resolved fluorescence microscopy using two-photon excitation

We have developed a high sensitivity time-resolved two-photon scanning microscope. At an excitation wavelength of 960 nm, a spatial point spread function of 0.3 μm (FWHM) radially and 0.9 μm (FWHM) axially is measured for an 1.25 N.A. objective. The light source is a mode-locked titanium-sapphire laser. The time resolution is 400 ps with common chromophores used in microscopy. Time resolution is obtained using the frequency-domain heterodyning technique in which the laser is synchronized at a very high cross-correlation frequency to the rest of the electronics. We demonstrate spatial and time resolution using well-characterized fluorescent microspheres. We show two applications of two-photon time-resolved fluorescence microscopy: time-resolved imaging of multiple dye labeled cells and quantitative cellular calcium concentration using a lifetime indicator.

Time‐resolved fluorescence microscopy using two‐photon excitation

We have developed a high sensitivity time-resolved two-photon scanning microscope. At an excitation wavelength of 960 nm, a spatial point spread function of 0.3 μm (FWHM) radially and 0.9 μm (FWHM) axially is measured for an 1.25 N.A. objective. The light source is a mode-locked titanium-sapphire laser. The time resolution is 400 ps with common chromophores used in microscopy. Time resolution is obtained using the frequency-domain heterodyning technique in which the laser is synchronized at a very high cross-correlation frequency to the rest of the electronics. We demonstrate spatial and time resolution using well-characterized fluorescent microspheres. We show two applications of two-photon time-resolved fluorescence microscopy: time-resolved imaging of multiple dye labeled cells and quantitative cellular calcium concentration using a lifetime indicator.

Transmembrane sodium energy gradient and calcium content in the adductor muscle of Mytilus edulis L. in relation to the toxicity of oil and organic chemicals

The electrochemical potential difference of sodium (sodium gradient) across cell membranes is the energy source of a number of important cellular functions, and failure to maintain the gradient is likely to affect these functions. The present investigation shows that the normal value of the gradient in the posterior adductor muscle of the blue mussel is about 10 000 J/mol, but a number of organic chemical compounds (oil, oil dispersants, formaldehyde and benzene) cause the value to drop. Subsequently, these compounds also lead to death among the animals. The results show furthermore that the mussels tolerate a moderate reduction of the sodium gradient, and that after moderate reductions the gradient returns to its normal value when the animals are transferred to clean sea water. However, if the sodium gradient drops below a value of about 6000 J/mol, the reversibility of the gradient depression seems to be lost, in that the sodium gradient in this case continues to fall even when the animals are transferred to clean seawater. In this situation the animals are moribund. The reduction in the sodium gradient is accompanied by a marked increase in the cellular content of calcium, which may be the direct cause of cellular death. The transmembrane sodium gradient and the cellular calcium content may have an interesting application as parameters for ecologically relevant evaluation of the toxicity of environmental pollutants.

Desensitization of the response to thyrotropin-releasing hormone in Xenopus oocytes is an amplified process that precedes calcium mobilization

Consecutive challenges with thyrotropin-releasing hormone (TRH) of oocytes expressing the TRH receptor (TRH-R) resulted in a pronounced desensitization, manifested as a decrease in chloride current amplitude and an increase in response latency. Exposure to low concentrations of TRH resulted in a marked decrease in the amplitude of the subsequent response to a higher concentration of the agonist, even though the second challenge was given before the onset of the response to the first challenge (within 3 - 15 s). Cellular calcium concentration ([Ca]i) did not increase within this interval, suggesting that calcium was not involved in the desensitization process. The latency of the second response, however, was either unchanged or shortened, implying additive effects of processes initiated by the first challenge. A longer interval (30 s) between the two challenges brought about a more pronounced decrease in amplitude and a prolongation of response latency. The calcium mobilization initiated by a second challenge with a high concentration of the agonist exhibited a longer latency, a lower rate of [Ca]i increase and a lower amplitude. Stimulation of co-expressed cholinergic-muscarinic ml receptors with a low concentration of acetylcholine resulted in a pronounced desensitization of the TRH response (heterologous desensitization). Activation of protein kinase C by beta-phorbol 12-myristate, 13-acetate resulted in a dose-dependent inhibition of the response to TRH, suggesting that protein kinase C was involved in desensitization. Chelerythrine, a specific inhibitor of protein kinase C, abolished a large part of the desensitization. A mutant of the TRH-R that lacks protein kinase C consensus phosphorylation sites in the C-terminal region, exhibited desensitization.(ABSTRACT TRUNCATED AT 250 WORDS)