No change in the density of the serotonin1A receptor, the serotonin4 receptor or the serotonin transporter in the dorsolateral prefrontal cortex from subjects with schizophrenia

Changes in serotonin receptors and the serotonin transporter have been reported in the dorsolateral prefrontal cortex from subjects with schizophrenia, an area of the brain thought to be important in the pathology of the illness. To further our understanding on how such changes could play a role in the pathology of the illness, in situ radioligand binding with autoradiography was used to measure the density of the serotonin1A receptor, the serotonin4 receptor and the serotonin transporter in the dorsolateral prefrontal cortex, obtained at autopsy, from 10 schizophrenic and 10 control subjects. The binding of [3H]8-OH-DPAT to serotonin1A receptor, [3H]GR113808 to the 5HT4 receptor and [3H]citalopram to serotonin transporter was not altered in subjects with schizophrenia. significantly, only in tissue from the control subjects was there a relationship between age and the density of the serotonin4 receptor in Brodmann's areas 8 (r = 0.71, P = 0.02) and 10 (r = -0.67, P = 0.03). Importantly, this confounding factor did not influence the comparison of the density of serotonin4 receptor in the tissue from the schizophrenic and control subjects. This study has failed to show a difference in the density of serotonin1A receptor, the serotonin4 receptor or the serotonin transporter in the dorsolateral prefrontal cortex (Brodmann's areas 8, 9 and 10) from subjects with schizophrenia. These data suggest that not all serotonergic markers are altered in the dorsolateral prefrontal cortex from schizophrenic subjects.     

Role of Phote-HrTH (Phormia terraenovae hypertrehalosemic hormone) in modulating the supercontractile muscles of the crop of adult Phormia regina Meigen

Phote-HrTH (Phormia terraenovae hypertrehalosemic hormone) has been demonstrated in the Diptera to be involved in flight metabolism, reproduction, and diapause. Each of these events needs the hormone’s action and requirement for carbohydrates is the common denominator. In Diptera, carbohydrates are taken up during feeding by action of the cibarial pump and are then stored in the crop. Using adult Phormia regina, both a bioassay and electrophysiological recordings show that Phote-HrTH slows down or inhibits the crop lobe muscles (P5) and, at the same time, stimulates the muscles of the pump 4 (P4) involved in pushing fluids out of the crop and up into the midgut for digestion. The EC₅₀ for P4 was in the nanomolar range while the IC₅₀ for P5 was 1.4–75.1 pM. The effect of Phote-HrTH on P4/5 suggests that the peptide is important in coordinating the two pumps, which are involved in moving carbohydrates up into the midgut for digestion. The adult crop organ is an essential storage organ for carbohydrates and now should be considered an important structure capable of delivering nutrients to the midgut for digestion.     

Effect of age, vitamin D, and calcium on the regulation of rat intestinal epithelial calcium channels

Transepithelial transport of calcium involves uptake at the apical membrane, movement across the cell, and extrusion at the basolateral membrane. Active vitamin D metabolites regulate the latter two processes by induction of calbindin D and the plasma membrane ATPase (calcium pump), respectively. The expression of calbindin D and the calcium pump declines with age in parallel with transepithelial calcium transport. The apical uptake of calcium is thought to be mediated by the recently cloned calcium channels-CaT1 (or ECaC2, TRPV6) and CaT2 (or ECaC1, TRPV5). The purpose of these studies was to determine whether there were age-related changes in intestinal calcium channel regulation and to identify the dietary factors responsible for their regulation. Young (2 months) and adult (12 months) rats were fed either a high calcium or low calcium diet for 4 weeks. The low calcium diet significantly increased duodenal CaT1 and CaT2 mRNA levels in both age groups, but the levels in the adult were less than half that of the young. The changes in calcium channel expression with age and diet were significantly correlated with duodenal calcium transport and with calbindin D levels. To elucidate the relative roles of serum 1,25(OH)2D3 and calcium in the regulation of calcium channel expression, young rats were fed diets containing varying amounts of calcium and vitamin D. Dietary vitamin D or exogenous 1,25(OH)2D3 more than doubled CaT1 mRNA levels, and this regulation was independent of dietary or serum calcium. These findings suggest that the apical calcium channels, along with calbindin and the calcium pump, may play a role in intestinal calcium transport and its modulation by age, dietary calcium, and 1,25(OH)2D3.     

Dependence of cardiac sarcoplasmic reticulum calcium pump activity on the phosphorylation status of phospholamban.

The application of electrophoretic resolution of the different phosphorylation species of pentameric phospholamban as a measure of phosphorylation stoichiometry was examined and verified. This enabled a critical evaluation of a number of issues central to current models of calcium pump regulation in cardiac sarcoplasmic reticulum. The phospholamban content of numerous preparations was calculated from 32P incorporation at a given stoichiometry, and compared with the respective calcium pump concentration (derived by comparison with a Coomassie-stained calibration curve of the fast-twitch skeletal muscle isozyme). A relationship of 2 mol of phospholamban:1 mol of ATPase resulted (phospholamban monomer:ATPase monomer), which was maintained throughout all vesicle subpopulations. The precise mechanism of coupling of phospholamban phosphorylation to calcium pump stimulation was probed, with particular emphasis on the individual contributions of each phosphorylated species (P1 to P5). This relationship could be adequately explained in three ways: (i) each phosphorylation event contributed equally to calcium pump stimulation; (ii) P1 and P2 were incapable of stimulating calcium pump activity, but full stimulation occurred upon generation of species P3; or (iii) the phosphospecies P1 was without effect on basal calcium pump activity, but successive phosphorylations contributed equally to stimulation. Finally, the functional implication of dual site phosphorylation of phospholamban (cAMP- and the endogenous calmodulin-dependent kinases) was examined. No change in calcium pump activity accompanied the second tier of phosphorylation over that achieved by the first.     

Modulation by polyelectrolytes of canine cardiac microsomal calcium uptake and the possible relationship to phospholamban

Calcium uptake and (Ca2+ + Mg2+)-ATPase activity in canine cardiac microsomes were found to be stimulated by heparin and various other polyanions. Prior treatment of the microsomes with the ionophores alamethicin or A23187 produced no change in the extent of stimulation of the ATPase activity by heparin yet eliminated net calcium uptake. This finding and a lack of change in the stoichiometric ratio of mol of calcium transported/mol of ATP hydrolyzed (calcium:ATP) suggest that the effect of heparin is on the calcium pump rather than on a parallel calcium efflux pathway. Certain polycationic compounds including poly-L-arginine and histone inhibited both cardiac and fast skeletal muscle microsomal calcium uptake and also produced no change in the stoichiometric ratio of calcium to ATP. Several lines of evidence indicate that the polyanionic compounds tested stimulate calcium uptake by interacting with phospholamban, the putative phosphorylatable regulator of the cardiac sarcoplasmic reticulum calcium pump, whereas polycationic compounds appear to interact with the pump. (i) Heparin stimulated calcium uptake to the same extent as protein kinase A or trypsin, whereas prior phosphorylation or tryptic cleavage of phospholamban from the membrane abolished the stimulatory effect of heparin. (ii) Calcium uptake and (Ca2+ + Mg2+)-ATPase activity in fast skeletal muscle microsomes, which lack phospholamban, were unaffected by heparin. (iii) Purified cardiac (Ca2+ + Mg2+)-ATPase activity was no longer stimulated by heparin yet was still inhibited by polycationic compounds. The heparin-induced stimulation of calcium uptake was dependent on the pH and ionic strength of the heparin-containing preincubation medium, hence electrostatic interactions appear to play a significant role in heparin's stimulatory action. The data are consistent with an inhibitory role of the positively charged cytoplasmic domain of phospholamban with respect to calcium pump activity and the relief of the inhibition upon reduction in phospholamban's positive charge by phosphorylation or binding of polyanions.     

Effect of prolactin on blood pressure and cardiovascular responsiveness in the rat

The effects of chronic ovine PRL (oPRL) infusion on resting systolic blood pressure (BP), heart rate, and pressor responsiveness to acute administration of norepinephrine and angiotensin were studied in adult male Sprague-Dawley rats. oPRL was administered over 7 days, via osmotic pump implanted ip on Day 1, at rates of 0, 0.15, 0.30, 0.60, 1.20, and 4.80 micrograms/hr. Resting BP and heart rate were indirectly determined in conscious rats by tail cuff technique on Days 1, 4, and 7 following pump implantation. In addition, acute pressor responses to ia norepinephrine (4.3 micrograms) and angiotensin (1.25 micrograms) were directly measured via arterial cannula in halothane-anesthetized rats on Day 7 of oPRL administration. oPRL infusion did not alter resting BP or heart rate over the 7 days. However, oPRL increased the BP response to norepinephrine at infusion rates of 0.60 and 4.80 micrograms/hr (P less than 0.01 vs controls). Body weight increases during the study were also greater in groups receiving 0.15, 0.30, 0.60, and 4.80 micrograms oPRL/hr (P less than 0.05) than those in control animals. oPRL decreased pressor responses to angiotensin at infusion rates of 0.30 and 1.20 micrograms/hr (P less than 0.01). These data suggest that, although the vascular effects of oPRL may not be evident under resting conditions, oPRL enhances vascular reactivity to norepinephrine infusion and depresses vascular reactivity to angiotensin infusion. Furthermore, at oPRL infusion rates which affect pressor responses to norepinephrine, oPRL increases body weight gain. These findings support a role for PRL in cardiovascular regulation during conditions of altered sympathetic activity.     

Insulin action on cardiac glucose transport. Studies on the role of the Na+/K+ pump

Isolated muscle cells from adult rat heart have been used to study the relationship between myocardial glucose transport and the activity of the Na+/K+ pump. 86Rb+-uptake by cardiac cells was found to be linear up to 2 min with a steady-state reached by 40-60 min, and was used to monitor the activity of the Na+/K+ pump. Ouabain (10(-3) mol/l) inhibited the steady-state uptake of 86Rb+ by more than 90%. Both, the ouabain-sensitive and ouabain-insensitive 86Rb+-uptake by cardiac cells were found to be unaffected by insulin treatment under conditions where a significant stimulation of 3-O-methylglucose transport occurred. 86Rb+-uptake was markedly reduced by the presence of calcium and/or magnesium, but remained unresponsive towards insulin treatment. Inhibition of the Na+/K+ pump activity by ouabain and a concomitant shift in the intracellular Na+ :K+ ratio did not affect basal or insulin stimulated rates of 3-O-methylglucose transport in cardiac myocytes. The data argue against a functional relationship between the myocardial Na+/K+ pump and the glucose transport system.     

Patterns of sarcomere activation, temperature dependence, and effect of ryanodine in chemically skinned cardiac fibers

Functionally skinned and electrochemically shunted myocytes were prepared by perfusing rat hearts with collagenase in order to obtain a technically improved measurement of sarcomere dynamics and to evaluate the role of sarcoplasmic reticulum in situ with respect to contractile activation. In the presence of micromolar calcium, the myocytes exhibited phasic and propagated contraction waves beginning at one end and proceeding along the myocyte. Beating rates, the propagation velocity of the activation wave, and single sarcomere shortening and relaxation velocities were obtained by manual or automated analysis of 16-mm film recorded at 170 frames/s from a camera attached to a microscope that was equipped with a temperature-controlled stage. In parallel experiments, calcium accumulation by the sarcoplasmic reticulum of the myocytes in situ was measured by direct isotopic tracer methods. The frequency (10-38 min-1) of spontaneous contractions, the velocity (1.9-7.4 microns . s-1) of sarcomere shortening, and the velocity (1.7-6.8 microns . s-1) of sarcomere relaxation displayed identical temperature dependences (Q10 = 2.2), which are similar to that of the calcium pump of sarcoplasmic reticulum and are consistent with a rate limit imposed by enzyme-catalyzed mechanisms on all these parameters. On the other hand, the velocity (77- 159 microns . s-1) of sequential sarcomere activation displayed a lower temperature dependence (Q10 = 1.5), which is consistent with a diffusion-limited and self-propagating release of calcium from one sarcomere to the other. The phasic contractile activity of the dissociated myocytes was inhibited by 10(-8)-10(6) M ryanodine (and not by myolemmal calcium blockers) under conditions in which calcium accumulation by sarcoplasmic reticulum in situ was demonstrated to proceed optimally. The effect of ryanodine is attributed to an interaction of this drug with sarcotubular structures, producing inhibition of calcium release from the sarcoplasmic reticulum. The consequent lack of sarcomere activation underlines the role of sarcoplasmic reticulum uptake and release in the phasic contractile activation of the electrochemically shunted myocytes.     

Calcium uptake of a rat liver microsomal subcellular fraction in response to in vivo administration of carbon tetrachloride.

ATP-dependent calcium uptake of rat liver microsomes is examined following ingestion of CC14 (2.5 ml/kg). Within 30 min there is an abrupt drop in calcium uptake activity of the liver microsomes. This activity remains down for 48 hours before slowly returning to normal levels. The effect is specific for CC14 as contrasted with CHC13 and CH2Cl2. The CCl4 does not affect similar calcium uptake activity of kidney microsomes. Calcium uptake activity of the liver mitochondria is unaffected. The first 12 hours after CCl4 ingestion there is a relatively slow rise in the calcium content of the liver tissue and mitochondria. After 12 hours a much larger influx of calcium into the tissue and the mitochondria takes place. Forty-eight hours after CCl4 ingestion the process begins to slowly reverse. The following postulated sequence may relate to the CCl4 hepatotocicity. CCl4 is activated to free radicals by the liver endoplasmic reticulum. The free radical inactivate calcium pump activity of the liver endoplasmic reticulum. Calcium levels of the cytoplasm increase and significantly modify ion permeability of the plasma membrane. High levels of external calcium enter the cytoplasm and are sequestered in the mitochondria. The high level of mitochondrial calcium uptake inhibits mitochondrial oxidative phosphorylation. The specific sensitivity of the calcium pump activity of liver microsomes to CCl4 further establishes the identity of a system seperate from the mitochondrial system. The above postulated sequence of events would suggest a critical role in liver metabolism for calcium pump activity of the endoplasmic reticulum.     

Functional domains of the in situ red cell membrane calcium pump revealed by proteolysis and monoclonal antibodies. Possible sites for regulation by calpain and acidic lipids

The functional domains of the in situ red cell membrane calcium pump were mapped by a double labeling technique. In inside-out vesicles (IOVs) the calcium pump was phosphorylated by [gamma-32P]ATP, the proteins blotted onto nitrocellulose and tagged by monoclonal antibodies raised against the purified pump protein. After proteolytic treatment of the IOVs by trypsin, chymotrypsin, or calpain-I, the fragmentation pattern of the enzyme was followed on the double-labeled immunoblots. The changes in the kinetics of the pump were examined by parallel measurements of the active calcium uptake in IOVs. By analysis of the results of tryptic digestion, it was possible to show that the antibodies recognized three different domains of the pump: 1) a Mr = 10,000-15,000 fragment (not seen directly) which includes the calmodulin-binding domain, 2) a nonphosphorylated Mr = 35,000 tryptic fragment, and 3) a phosphorylated fragment of Mr = 76,000-81,000. Chymotrypsin or calpain-I digestion of the membranes produced one major, Mr = 125,000 fragment, which had lost antibody-binding region 1. Production of this fragment coincided with the loss of calmodulin dependence and with a calmodulin-like activation of IOV calcium uptake (high Vmax, cooperativity in calcium activation). The Mr = 125,000 fragment was further activated by acidic lipids producing high Vmax and low K 1/2 (Ca2+) with no cooperativity. Based on these data a kinetic model and a functional map of the plasma membrane calcium pump is suggested.     

Lack of effect of corticotropin releasing factor on hypothalamic dopamine and serotonin synthesis turnover rates in rats

Catecholamine and serotonin neurons in the hypothalamus regulate the secretion of corticotropin releasing factor (CRF). We considered the possibility that CRF might in turn affect the activity of these aminergic neurons. We examined the effect of intracisternal administration of synthetic CRF on the synthesis turnover rates of dopamine and serotonin in the hypothalamus of adult male rats using two different methods to assess turnover. In one study, we measured the accumulation of L-dihydroxyphenylalanine (L-DOPA) or 5-hydroxytryptophan (5-HTP) in mediobasal hypothalamus after L-aromatic amino acid decarboxylase inhibition with m-hydroxybenzylhydrazine 20 min before sacrifice, and in the second study we measured the accumulation of dopamine, norepinephrine, epinephrine and serotonin after monoamine oxidase inhibition with pargyline 20 min before sacrifice. The commercial CRF which we administered intraarterially increased plasma ACTH and corticosterone concentrations. Intracerebral CRF 5 to 20 micrograms 20 min before sacrifice or 20 micrograms 110 min before sacrifice did not alter the m-hydroxybenzylhydrazine-induced accumulation of L-DOPA or 5-HTP when compared with saline vehicle-injected controls. CRF 20 micrograms did not alter basal concentration or pargyline-induced accumulation of the catecholamines or serotonin in whole hypothalamus when compared with saline vehicle-injected controls. Thus, intracisternal administration of CRF did not alter hypothalamic dopamine or serotonin synthesis rates as assessed by two nonsteady state turnover methods. The data suggest that the release of CRF from neurons in hypothalamus does not alter the activity of catecholamine or serotonin neurons in the hypothalamus of normal adult male rats.     

Effect of Tetrahydrobiopterin on Serotonin Synthesis, Release, and Metabolism in Superfused Hippocampal Slices

The effects of 6R-5,6,7,8-tetrahydro-L-biopterin (6R-BH4), the in vivo cofactor for tryptophan hydroxylase, on the synthesis, release, and metabolism of serotonin were studied in superfused slices from rat hippocampus. 6R-BH4 did not alter the spontaneous release of [3H]serotonin but it did significantly increase release when slices were depolarized with 30 mM KCl. Under the same incubation conditions, 6R-BH4 altered neither the synthesis (basal or tryptophan-stimulated) nor the metabolism of serotonin in hippocampal slices. The synthetic pteridine 6-methyl-5,6,7,8-tetrahydropterin also augmented release under depolarizing conditions whereas biopterin, the oxidized form of 6R-BH4, did not. The 6S isomer of BH4, which is relatively inactive as a cofactor for tryptophan hydroxylase, was equipotent with 6R-BH4 in stimulating serotonin release. 6R-BH4 did not inhibit serotonin uptake nor did it function as a serotonin autoreceptor antagonist to increase release. A direct serotonin releasing effect of 6R-BH4, like that produced by p-chloroamphetamine, could also be ruled out. At suboptimal concentrations of extracellular calcium, the KCl-induced release of 3H was significantly reduced, yet the increase in release caused by BH4 remained the same in magnitude. It is concluded that 6R-BH4 increases the depolarization-induced release of serotonin through an interaction with the release mechanism itself, possibly by enhancing calcium influx or by increasing the sensitivity of the release mechanism to calcium. The effects of 6R-BH4 on serotonin release are independent from its function as the cofactor for tryptophan hydroxylase.     

The possible roles of the monoaminergic system in the feeding of the snail Helix pomatia

The possible role of serotonin and dopamine in the feeding of Helix pomatia was studied applying immunocytochemical, biochemical, and behavioral techniques as well as bioassay experiments. Immunocytochemistry showed that dopamine-containing (thyrosin-hydroxylase-immunoreactive) neuronal elements of the crop and the gizzard belong to the intrinsic part, whereas serotonin-containing (serotonin-immunoreactive) neuronal elements belong to the extrinsic part of the gastrointestinal nervous system. Bioassay studies on the spontaneous contractions of the crop and the gizzard showed that dopamine affected only the longitudinal muscle contractions by increasing both the tonus and contractility, whereas serotonin was effective on both the longitudinal and circular muscle contractions. Serotonin increased the tonus and contractility of longitudinal muscles in the crop but decreased them in the gizzard. Serotonin decreased the tonus and contractility of the circular muscles in the crop but increased them in the gizzard. Serotonin effects on the circular muscle of the gizzard were concentration dependent between a range of 10(-5) M-3 x 10(-5) M. HPLC measurements of monoamines in starved and satiated animals showed that the concentration of both dopamine and serotonin significantly decreased in both the CNS and different parts of the gastrointestinal tract of satiated animals, suggesting a significant monoamine liberation during feeding. The injection of monoamines (10(-3) and 10(-2) M) into the body cavity of starved animals showed that only dopamine was able to induce feeding whereas serotonin increased the general activity of the animals suggesting that the initiation of feeding is rather dopamine than serotonin dependent.     

The salivary and crop apyrase activity of Rhodnius prolixus

A calcium dependent apyrase activity (ATP→AMP + 2Pi) has been characterized in the salivary secretion of Rhodnius prolixus. High levels of this activity were found in the crop of all stages of larvae and the adults after a single blood or saline meal. The activity persisted for several days but was totally absent in the crop insects from which the salivary glands had been removed. The use of this activity as a saliva marker shows that the insect salivates during the whole meal and most of the saliva is ingested with the food. The physiological role of this activity is discussed. A simple method for saliva collection and a technique for the surgical ablation of the salivary glands in adult insects are described.     

The calcium ionophore A23187 is a potent stimulator of the vitamin D3-25 hydroxylase in hepatocytes isolated from normocalcaemic vitamin D-depleted rats.

The role played by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and/or by calcium on the C-25 hydroxylation of vitamin D3 (D3) was studied in hepatocytes isolated from D-depleted rats which were divided into four treatment groups: Group 1 served as controls, Group 2 received calcium gluconate, Groups 3 and 4 were infused with 1,25(OH)2D3 at 7 and 65 pmol/24 h x 7 days respectively. The treatments normalized serum calcium in all but the controls which remained hypocalcaemic, while serum 1,25(OH)2D3 remained low in Groups 1 and 2 but increased to physiologic and supraphysiologic levels in Groups 3 and 4. The data show that basal D3-25 hydroxylase activities were not significantly affected by any of the treatments. Addition of CaCl2, EGTA, or Quin-2 in vitro revealed that relative to basal values, EGTA strongly inhibited the enzyme activity in all groups (P less than 0.0001), except in G 1; Quin-2 and CaCl2 had no significant effect on the activity of the enzyme in any of the groups. Addition of 1,25(OH)2D3 or A23187 in vitro in the presence of CaCl2 revealed that 1,25(OH)2D3 did not significantly affect enzyme activity, while A23187 was found to stimulate its activity in vitamin D-depleted animals, but most specifically in Group 2 (P less than 0.001); low serum calcium (Group 1) dampened (P less than 0.01), and 1,25(OH)2D3 treatment in vivo totally blunted (P less than 0.001) the response to A23187. The data suggest that 1,25(OH)2D3 supplementation in vivo has per se little or no effect on the basal D3-25 hydroxylase activity. The data show, however, that the magnitude of the response to various challenges in vitro is greatly influenced by the conditioning in vivo of the animals. They also show that A23187 can be a potent stimulator of the enzyme activity, which allowed us to demonstrate a significant reserve for the C-25 hydroxylation of D3 which is well expressed in hepatocytes obtained from D-depleted calcium-supplemented rats.     

Effects of neuroactive substances on the activity of subcommissural organ cells in dispersed cell and explant cultures

The subcommissural organ (SCO), an ependymal (glial) circumventricular organ, releases glycoproteins into the cerebrospinal fluid; however, the regulation of its secretory activity is largely unknown. To identify neuroactive substances that may regulate SCO activity, we investigated immunocytochemically identified bovine SCO cells by means of calcium imaging. This analysis was focused on: (1) serotonin (5HT) and substance P (SP), immunocytochemically shown to be present in axons innervating the bovine SCO; and (2) ATP, known to activate glial cells. 5HT had no effect on the intracellular calcium concentration ([Ca(2+)](i)), and its precise role remains to be clarified. SP elicited rises in [Ca(2+)](i) in approx. 30% and ATP in even 85% of the analyzed SCO cells. These effects were dose-dependent, involved NK(3) and P2Y(2) receptors linked to G protein and phospholipase C (PLC) activation, and could not be mimicked by forskolin or 8-bromo-cAMP. In 50% of the SP-sensitive cells, the increases in [Ca(2+)](i) comprised calcium release from thapsigargin-sensitive intracellular stores and an influx of extracellular calcium via protein kinase C (PKC)-induced opening of L-type voltage-gated calcium channels (VGCCs). In the remaining SP-sensitive cells, the increase in [Ca(2+)](i) was caused exclusively by influx of extracellular calcium via VGCCs of the L-type. In all ATP-sensitive cells the increase in [Ca(2+)](i) involved calcium release from thapsigargin-sensitive intracellular stores and a PKC-mediated influx of extracellular calcium via L-type VGCCs. Our data suggest that SP and ATP are involved in regulation of the activity of SCO cells.     

Serotonin stimulates calcium influx in isolated rat adrenal zona glomerulosa cells.

To investigate the role of calcium as a second messenger in serotonin-stimulated aldosterone secretion, radiolabelled calcium influx studies were carried out in purified rat adrenal zona glomerulosa cells using 45CaCl2. The results show that serotonin caused calcium influx within 45 seconds of addition and this continued for up to 105 seconds. Angiotensin II also caused calcium influx; however, the effect was significantly smaller than that of serotonin. Serotonin-stimulated calcium influx could be inhibited by the calcium antagonist verapamil and by methysergide, a selective serotonin receptor type-1/2 antagonist. The data indicate that serotonin directly stimulates calcium uptake in zona glomerulosa cells via calcium channels which are coupled to specific serotonin receptors.     

Calcium transport in transverse tubules isolated from rabbit skeletal muscle

Isolated transverse tubule vesicles free of sarcoplasmic reticulum transport calcium with high affinity in the presence of ATP. The calcium transport by transverse tubules differs from calcium transport by sarcoplasmic reticulum. It is not increased by oxalate or phosphate, it has a different temperature dependence, it is inhibited by sub-micromolar concentrations of orthovanadate, it is stimulated by calmodulin, and is inhibited by quercetin without causing calcium release. The rates of calcium transport by transverse tubules are two orders of magnitude lower than those of sarcoplasmic reticulum, suggesting that the calcium pump protein of transverse tubules is a minor component of the membrane. Addition of calmodulin to transverse tubule vesicles--treated with high salt in the presence of EGTA to remove endogenous calmodulin--caused a marked stimulation of transport rates at low concentrations of calcium, and decreased from 1.0 to 0.3 microM the calcium concentration at which half-maximal rates of transport were obtained. A role for the transverse tubule calcium pump in maintaining low sarcoplasmic calcium concentrations is proposed.     

Blockage of a pump-related calcium-efflux pathway in light sarcoplasmic reticulum vesicles by Mops

Mops, used as a proton buffer, specifically enhances the accumulation of calcium or strontium by light sarcoplasmic reticulum vesicles driven by ATP or dinitrophenylphosphate as energy-yielding substrates when calcium-precipitating agents are absent. The enhancement of ion uptake by Mops is much greater for strontium than for calcium and is further increased when potassium is replaced by sodium as the dominant monovalent cation. Mops affects neither the activity of the calcium- or strontium-activated transport enzyme nor the active accumulation of calcium in the presence of oxalate, i.e. when the pump runs unidirectionally forward. Passive calcium and strontium efflux rates of approximately 40-50 nmol.mg-1.min-1 are considerably reduced when histidine/glycerophosphate or Tris/maleate are exchanged for Mops. The observed passive efflux rates and their modulation by Mops are too small, in relation to the rate of ion influx, to account for either the relatively small calcium and strontium load in the absence of precipitating agents or for its modulation by Mops. The results imply that the pump itself mediates ion efflux dependent on pump activity and the different degree of saturation of lumenal ion-binding sites by calcium and strontium, as well as their susceptibility to Mops.