Long openings of calcium channels in fetal rat ventricular cardiomyocytes

Whole-cell and single-channel Ca²⁺ currents (I_Ca) of single fetal (12 – 18 days) and neonatal (1 – 10 d) rat ventricular myocytes were recorded using patch clamp techniques. Whole-cell I_Ca density increased markedly during the fetal period and remained almost constant during the neonatal period. In cell-attached patch recordings (with 110 mM Ba²⁺ in the pipette), the L-type Ca²⁺ channels, observed on both fetal d-12 and neonatal d 5, had the same conductance (23 pS). On fetal d-12, many relatively long openings were observed in addition to brief openings, whereas on neonatal d-5, long openings were less observed and brief openings dominated. Therefore, long openings of the Ca²⁺ channels occur in early development in rat heart cells, similar to that reported for chick hearts [7, 8]. The increase of the whole-cell current amplitude observed during development may be due to an increase in available channel number.     

Effects of endurance training on intracellular calcium concentration in T lymphocytes

The purpose of the present study was to determine whether 12 months of endurance training reduced [Ca²⁺]i in T helper (CD4⁺) lymphocytes in trained (TR) men compared to untrained (UT). Fourteen trained (Ironman triathletes) and nine untrained (sedentary) men volunteered for the study. The TR group averaged 12 km of swimming, 300 km of cycling and 60 km of running per week during the year. Resting blood samples were taken from TR (VO_2peak 64 ± 2 ml kg⁻¹ min⁻¹) and UT (VO_2peak 42 ± 2 ml kg⁻¹ min⁻¹) subjects every 4 weeks for 52 weeks (October 1, 1999–October 1, 2000). Leukocyte concentration was measured using a full blood count. Unstimulated CD4⁺ lymphocytes were separated and analysed for changes in free ([Ca²⁺]i) and total ([Ca²⁺]t) calcium using flow cytometry. There were no significant differences in leukocyte concentration between UT and TR groups. There were significant differences between TR and UT in [Ca²⁺]i (October B and November), and [Ca²⁺]t (January and March). There were also significant sequential monthly changes in both [Ca²⁺]i and [Ca²⁺]t for TR and UT groups during the study. Significant increases in [Ca²⁺]i and [Ca²⁺]t during summer (January and March) for both TR and UT groups suggest an increase in intracellular signalling during hot weather. [Ca²⁺]i and [Ca²⁺]t were significantly lower in TR lymphocytes during November and March, suggesting that endurance training during warmer months may decrease [Ca²⁺]i through altered intracellular signalling, possibly to maintain lymphocyte function during heat stress.     

Effect of internal calcium concentration on calcium currents in rat sensory neurones

Using the patch-clamp technique in whole-cell configuration we have investigated the effect of increasing the internal calcium concentration (Ca_i) from below 10^–8 M to 10^–6 M on the three calcium currents: I_{Ca, T} (T for transient), I_{Ca, S} (S for sustained), I_{Ca, N} (N for neither), recently described in rat sensory neurones> Increasing Ca_i led to a dose-dependent reduction of the amplitude of I_{Ca, S} and, as Ca_i reached 5×10^–7M I_{Ca, S} was nearly abolished. I_{Ca, N} is well evidenced from 5×10^–10 M to 10^–7 M where its is a large current. Preliminary observations indicate an increase of its inactivation rate following, as expected for a possible Ca_i dependent-inactivation, the increase of Ca_i from 5×10^–10 M to 10^–7 M. With Ca=5×10^–7 M, all the cells displayed I_{Ca, T} and half of the cells in addition I_{Ca, N}, but it was of small amplitude. At Ca_i=10^–6 M, most of the recorded cells only exhibited I_{Ca, T}.     

Effects of caffeine on intracellular calcium,calcium current and calcium-dependent potassium current in anterior pituitary GH3 cells

Caffeine elicits physiological responses in a variety of cell types by triggering the mobilization of Ca²⁺ from intracellular organelles. Here we investigate the effects of caffeine on intracellular Ca²⁺ concentration ([Ca²⁺]_i) and ionic currents in anterior pituitary cells (GH₃) cells. Caffeine has a biphasic effect on Ca²⁺-activated K⁺ current [I _K(Ca)]: it induces a transient increase superimposed upon a sustained inhibition. While the transient increase coincides with a rise in [Ca²⁺]_i, the sustained inhibition of I _K(Ca) is correlated with a sustained inhibition of the L-type Ca²⁺ current. The L-type Ca²⁺ current is also inhibited by other agents that mobilize intracellular Ca²⁺, including thyrotropin releasing hormone (TRH) and ryanodine, but in a matter distinct from caffeine. Unlike the caffeine effect, the TRH-induced inhibition washes-out under whole-cell patch-clamp conditions and is eliminated by intracellular Ca²⁺ chelators. Likewise, the ryanodine-induced inhibition desensitizes while the caffeine-induced inhibition does not. Simultaneous [Ca²⁺]_i and Ca²⁺ current measurements show that caffeine can inhibit Ca²⁺ current without changing [Ca²⁺]_i. Single-channel recordings show that caffeine reduces mean open time without affecting single-channel conductance of L-type channels. Hence the effects of caffeine on ion channels in GH₃ cells are attributable both to mobilization of intracellular Ca²⁺ and to a direct effect on the gating of L-type Ca²⁺ channels.     

Amitriptyline modulates calcium currents and intracellular calcium concentration in mouse trigeminal ganglion neurons

Migraine is increasingly recognized as a channelopathy, and abnormalities of voltage-activated ionic channels could represent the molecular basis for the altered neuronal functioning. The high-voltage-activated (HVA) Ca²⁺ channels in the trigeminovascular system play a role in the pathophysiology of migraine. In the present study, effects of amitriptyline (AMT), a commonly used migraine prophylactic drug, on the HVA calcium currents (I_Ca) were examined in mouse trigeminal ganglion neurons using whole-cell patch clamp technique. AMT produced concentration- and use-dependent inhibition of HVA I_Ca. Bath application of GÖ-6983 (a selective protein kinase C inhibitor) or H89 (a protein kinase A inhibitor) did not reduce the AMT-induced inhibition of HVA I_Ca. A similar inhibition was observed when calcium imaging was used to directly monitor the effects of AMT on KCl-induced increments of intracellular Ca²⁺ concentration ([Ca²⁺]_i). By blocking HVA Ca²⁺ channels and Ca²⁺ entry into cells, AMT could prevent the release of neurotransmitters and help restore the neuronal threshold for excitation. Our findings suggest interesting therapeutic mechanisms for AMT in migraine prevention.     

Dependence of intracellular free calcium and tension on membrane potential and intracellular pH in single crayfish muscle fibres

The dependence of intracellular free calcium ([Ca²⁺]_i) and tension on membrane potential and intracellular pH (pH_i) was studied in single isolated fibres of the crayfish claw-opener muscle using ion-selective microelectrodes. Tension (T) was quantified as a percentage of the maximum force, or as force per cross-sectional area (N/cm²). In resting fibres, pH_i had a mean value of 7.06. Contractions evoked by an increase extracellular potassium ([K⁺]₀) produced a fall in pH_i of 0.01–0.05 units. The lowest measured levels of resting [Ca²⁺]_i corresponded to a pCa_i (= –log [Ca²⁺]_i) of 6.8. Intracellular Ca²⁺ transients recorded during K⁺-induced contractions did not reveal any distinct threshold for force development. Both the resting [Ca²⁺]_i and resting tension were decreased by an intracellular alkalosis and increased by an acidosis. The sensitivity of resting tension to a change in pH_i (quantified as –dT/ dpH_i) showed a progressive increase during a fall in pH_i within the range examined (pH_i 6.2–7.5). The pH_i/[Ca²⁺]_i and pH_i/tension relationships were monotonic throughout the multiphasic pH_i change caused by NH₄Cl. A fall of 0.5–0.6 units in pH_i did not produce a detectable shift in the pCa_i/tension relationship at low levels of force development. The results indicate that resting [Ca²⁺]_i is slightly higher than the level required for contractile activation. They also show that the dependence of tension on pH_i in crayfish muscle fibres is attributable to a direct H⁺ and Ca²⁺ interaction at the level of Ca²⁺ sequestration and/or transport. Finally, the results suggest that in situ, the effect of pH on the Ca²⁺ sensitivity of the myofibrillar system is not as large as could be expected on the basis of previous work on skinned crustacean muscle fibres.     

Effects of intracellular Ca2+ chelating compounds on inward currents caused by Ca2+ release from sarcoplasmic reticulum in guinea-pig atrial myocytes

Ca²⁺ release from the sarcoplasmic reticulum (SR) of mammalian cardiac myocytes occuring either due to activation by a depolarization or the resulting transmembrane Ca²⁺ current (I _Ca), or spontaneously due to Ca²⁺ overload has been shown to cause inward current(s) at negative membrane potentials. In this study, the effects of different intracellular Ca²⁺ chelating compounds on I _Ca-evoked or spontaneous Ca²⁺-release-dependent inward currents were examined in dialysed atrial myocytes from hearts of adult guinea-pigs by means of whole-cell voltage-clamp. As compared to dialysis with solutions containing only a low concentration of a high affinity ethylene glycol-bis(-aminoethylether) N,N,N,N-tetraacetic acid (EGTA) like chelator (50–200 M), inward membrane currents (at –50 mV) due to evoked Ca²⁺ release, spontaneous Ca²⁺ release or Ca²⁺ overload following long-lasting depolarizations to very positive membrane potentials are prolonged if the dialysing fluid contains a high concentration of a low affinity Ca²⁺ chelating compound such as citrate or free adenosine 5-triphosphate (ATP). Without such a non-saturable Ca²⁺ chelator in the dialysing fluid, Ca²⁺-release-dependent inward currents are often oscillatory and show an irregular amplitude. With a low affinity chelator in a non-saturable concentration, discrete inward currents with constant properties can be recorded. We conclude that the variability in Ca²⁺-release-dependent inward current seen in single cells arises from spatial inhomogeneities of intracellular Ca²⁺ concentration ([Ca²⁺]_i) due to localized saturation of endogenous and exogenous high affinity Ca²⁺ buffers (e.g. [2]). This can be avoided experimentally by addition of a non-saturable buffer to the intracellular solution. This condition might be useful, if properties of Ca²⁺ release from the SR and/ or the resulting membrane current, like for example arrhythmogenic transient inward current, are to be investigated on the single cell level.     

The intracellular Ca2+ concentration optimal for T cell activation is quite different after ionomycin or CD3 stimulation

The relationship between the initial increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i) (measured at the single-cell level with an imaging system) and the ensuing proliferation was examined in a human T cell clone stimulated by a phorbol ester in combination with ionomycin, thapsigargin or an anti-CD3 mAb (monoclonal antibody against the CD3 molecule, UCHT1). From the responses to various ionomycin concentrations, one can define a range of [Ca²⁺]_i values (400–900 nM) which appears optimal for T cell proliferation; lower [Ca²⁺]_i values are suboptimal, higher values are cytotoxic. It was then examined if the [Ca²⁺]_i requirements were similar following anti-CD3 stimulation. [Ca²⁺]_i oscillations elicited by a concentration of UCHT1 (1/1,000) optimal for mitogenicity fall precisely within the 400–900 nM range. However, very low concentrations of UCHT1 (1/100,000) which evoke barely detectable [Ca²⁺]_i responses still cause the cells to proliferate. The possibility that the lower [Ca²⁺]_i requirements observed following anti-CD3 stimulation was due to [Ca²⁺]_i oscillations was tested under conditions which prevented the appearance of these oscillations. It turns out that an oscillatory Ca²⁺signal is not more mitogenic than a sustained augmentation of [Ca²⁺]_i. Finally, it was examined if overstimulation via CD3 could have toxic consequences similar to those elicited after ionomycin overstimulation. Large transient [Ca²⁺]_i responses can be observed following anti-CD3 stimulation in appropriate conditions, and namely in T cells pretreated with interleukin-2. These [Ca²⁺]_i augmentations are not cytotoxic. A role for the plasmalemmal Ca²⁺ pump in the prevention of cytotoxicity can be demonstrated. In conclusion, the correspondence between the [Ca²⁺]_i response and cell proliferation is entirely different following stimulation by ionomycin and by anti-CD3. In addition, cell proliferation evoked by very low UCHT1 concentration might reveal the existence of a yet unidentified activation pathway.     

Effect of contrast media on free calcium concentration in rat bloodin vivo

The X-ray contrast media omnipaque=melitrast=ultravist <peritrast=hexabrix<triombrast<bilignost decrease the concentration of free calcium ions in blood serum of “sensitive” Wistar ratsin vivo by 15–30%. The number of “sensitive” rats (33–67%) did not depend on the dose and type of the agents. The magnetic resonance imaging agent magnevist decreased serum calcium by 10% in all rats. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 11, pp. 542–545, November, 1998     

Effects of extracellular calcium concentration on neurite outgrowth in PC12 cells by staurosporine

Staurosporine as an inhibitor of protein kinases can induce neuronal differentiation in PC12 cells. We investigated the role of extracellular Ca²⁺ on staurosporine inducing neurite outgrowth in PC12 cells. The cells were cultured during cell differentiation in the presence of 214 nM staurosporine with 0.0–0.7 Ca²⁺mM as treatment media. We obtained the fraction of neurite-bearing cells, total neurite length and the percentage of cytotoxiciy. The results showed that decrease or increase of extracellular calcium ions resulted in correspondingly significant decrease or increase in total neurite length and cell differentiation in treated cells. With an increase of extracellular calcium concentration from 0.0 to 0.7 mM, the percentage of cytotoxicity of the PC12 cells decreased (p < 0.05). Our data suggest that staurosporine uses the extracellular calcium ions to affect on neurite outgrowth.     

Caffeine-induced calcium release from isolated sarcoplasmic reticulum of rabbit skeletal muscle

The essential conditions for the Ca²⁺ releasing action of caffeine from isolated sarcoplasmic reticulum (SR) of rabbits were evaluated by an investigation into the effects of Ca²⁺, Mg²⁺, MgATP^2–, and ATP concentration, ionic strength, and degree of loading. The heavy fraction (4,500×g) of the reticulum was used. Except for the study on degree of loading, 0.2 mg protein·ml^–1 SR was loaded actively with 0.02 mM⁴⁵CaCl₂, resulting in >90 nmol·mg protein^–1 at steady state, and then the effects of various parameters with or without (control) caffeine were tested.It was found that (1) caffeine induces a transient, dosedependent release of Ca²⁺, (2) the absolute amount of Ca²⁺ released by caffeine increases with the Ca²⁺ load of the SR, (3) increasing the ionic strength () from 0.09 to 0.3 lowers the threshold concentration of caffeine, (4) the SR is refractory to a repeated challenge by a caffeine concentration causing maximal effect, (5) caffeine-induced Ca²⁺ release increases with increasing (a) external Ca²⁺ concentrations up to 5 M total Ca²⁺ (or 3 M free Ca²⁺) and (b) free ATP concentrations up to 0.45 mM, and (6) caffeine-induced Ca²⁺ release is not affected by changes of either the Mg²⁺ or the MgATP^2– concentration.     

Measurement by Quin2 of changes of the intracellular calcium concentration in strips of the rabbit ear artery and of the guinea-pig ileum

Ca²⁺ transient and force development were investigated in smooth muscle strips of the rabbit ear artery and the longitudinal layer of the guinea-pig ileum by using the fluorescent indicator Quin2. Agonists only transiently increased the flourescence intensity despite the enhanced contraction while excess potassium resulted in a maintained light signal. In Ca²⁺ free solutions the release by an agonist of Ca²⁺ from an intracellular store can be demonstrated. These observations illustrate the usefullness of the Ca²⁺ indicator Quin2 in the study of the excitation-contraction coupling in smooth muscle under various conditions.     

Ghrelin increases intracellular Ca(2+) concentration in the various hormone-producing cell types of the rat pituitary gland

Ghrelin, isolated from the stomach as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), has potent growth hormone release ability in vivo and in vitro. Although GHS-R is abundantly expressed in the pituitary gland, there is no direct evidence of a relationship between hormone-producing cells and functional GHS-R in the pituitary gland. The aim of this study was to determine which anterior pituitary cells respond to ghrelin stimulation in male rats. We performed Fura-2 Ca(2+) imaging analysis using isolated pituitary cells, and performed immunocytochemistry to identify the type of pituitary hormone-producing cells. In Fura-2 Ca(2+) imaging analysis, ghrelin administration increased the intracellular Ca(2+) concentration in approximately 50% of total isolated anterior pituitary cells, and 20% of these cells strongly responded to ghrelin. Immunocytochemical analysis revealed that 82.9±1.3% of cells that responded to ghrelin stimulation were GH-immunopositive. On the other hand, PRL-, LH-, and ACTH-immunopositive cells constituted 2.0±0.3%, 12.6±0.3%, and 2.5±0.8% of ghrelin-responding pituitary cells, respectively. TSH-immunopositive cells did not respond to ghrelin treatment. These results suggest that ghrelin directly acts not only on somatotrophs, but also on mammotrophs, gonadotrophs, and corticotrophs in the rat pituitary gland.     

Modulation of single slow (L-type) calcium channels by intracellular ATP in vascular smooth muscle cells

Involvement of ATP in the regulation of slow (L-type) Ca²⁺ channels of vascular smooth muscle cells was investigated by recording single Ca²⁺ channel currents (single-channel conductance of 18 pS) using a patch clamp technique. In the cell-attached configuration, intracellular composition was modified by permeabilizing the cell membrane with mechanical disruption at one end of the cell. Single cells were freshly isolated from guinea-pig portal vein by collagenase treatment. For the channel recordings, the pipette solution contained 100 mM Ba²⁺ and the bath contained K⁺-rich solution (with 5 mM EGTA) to depolarize the membrane to near 0 mV. The channel activity decreased usually within 3 min after permeabilizing the cell end and exposure to ATP-free bath solution. If ATP (1–5 mM) was applied to the bath (access to cell interior) before complete disappearance of channel activity, channel activity was partially recovered. ATP did not change the current amplitude (i) or the mean open time of the channels, whereas the number of channels available for opening and/or the probability of their being open (NP _o) were increased by ATP. A non-hydrolyzable analogue of ATP, AMP-PNP, did not exert an ATP-like effect; ATP--S had a weak effect. With 1 M Bay-K-8644 (Ca²⁺ channel agonist) in the pipette, the activity of the Ca²⁺ channel was high; such activity persisted for more than 10 min after permeabilizing the cell and exposting to ATP-free solution containing KCN (1 mM) and 2-deoxy-d-glucose (10 mM). These results indicate that activation of slow Ca²⁺ channels requires ATP. The effect of ATP may be exerted by phosphorylation and/or an energy-requiring step. Bay-K-8644 may change the nature of the slow Ca²⁺ channel, making it resistant to rundown.     

Differences in magnesium and calcium effects on N-methyl-D-aspartate- and quisqualate-induced decreases in extracellular sodium concentration in rat hippocampal slices

Summary Decreases in extracellular sodium concentration ([Na⁺]o) and associated slow negative field potentials (fp's) were monitored with double barreled sodium sensitive/reference microelectrodes in area CA1 of rat hippocampal slices during iontophoretic application of the glutamate receptor agonists N-methyl-D-aspartate (NMDA) and quisqualate (quis). The effects of lowering [Ca²⁺]o on these signals were compared to those of lowering [Mg²⁺]o. Both NMDA- and quis-induced decreases in [Na⁺]o of up to 60 mM and in the fp's of up to 8 mV. Decreasing [Mg²⁺]o enhanced NMDA-induced signals, whereas quis-induced signals were unaffected. Lowering [Ca²⁺]o also enhanced NMDA signals, although somewhat less than lowering [Mg²⁺]o. This effect was still present, even when voltage dependent Na⁺ currents were blocked by 10^-7 tetrodotoxin. Interestingly, quis-induced signals could be enhanced in a low Ca²⁺ medium as well, but only when high quis concentrations were used. The results suggest that, during the sorts of large decreases of [Ca²⁺]o observed during seizure activity, activation of NMDA receptors is facilitated.Supported by a grant from the Deutsche Forschungsgemeinschaft (He 1128/2-4)     

The effect of L-type Ca2+ channel blockers on anoxia-induced increases in intracellular Ca2+ concentration in rabbit proximal tubule cells in primary culture

Ca²⁺ channel blockers (CCB) have been shown to be protective against ischaemic damage of the kidney, suggesting an important role for intracellular Ca²⁺ ([Ca²⁺]_i) in generating cell damage. To delineate the mechanism behind this protective effect, we studied [Ca²⁺]_i in cultured proximal tubule (PT) cells during anoxia in the absence of glycolysis and the effect of methoxyverapamil (D600) and felodipine on [Ca²⁺]_i during anoxia. A method was developed whereby [Ca²⁺]_i in cultured PT cells could be measured continuously with a fura-2 imaging technique during anoxic periods up to 60 min. Complete absence of O₂ was realised by inclusion of a mixture of oxygenases in an anoxic chamber. [Ca²⁺]_i in PT cells started to rise after 10 min of anoxia and reached maximal levels at 30 min, which remained stable up to 60 min. The onset of this increase and the maximal levels reached varied markedly among individual cells. The mean values for normoxic and anoxic [Ca²⁺]_i were 118±2 (n=98) and 662±22 (n=160) nM, respectively. D600 (1 M), but not felodipine (10 M), significantly reduced basal [Ca²⁺]_i in normoxic incubations. During anoxia 1 M and 100 M D 600 significantly decreased anoxic [Ca²⁺]_i levels by 22 and 63% respectively. Felodipine at 10 M was as effective as 1 M D600. Removal of extracellular Ca²⁺ and addition of 0.1 mM La³⁺ completely abolished anoxia-induced increases in [Ca²⁺]_i. We conclude that anoxia induces increases in [Ca²⁺]_i in rabbit PT cells in primary culture, which results from Ca²⁺ influx. Since this Ca²⁺ influx is partially inhibited by low doses of CCBs, Ltype Ca²⁺ channels may be involved.     

Effects of high-intensity training and acute exercise on in vitro function of rat sarcoplasmic reticulum

To evaluate the effects of high-intensity training and/or a single bout of exercise on in vitro function of the sarcoplasmic reticulum (SR), the rats were subjected to 8 weeks of interval running program (final training: 2.5-min running × 4 sets per day, 50 m/min at 10% incline). Following training, SR function, i.e., Ca²⁺-ATPase activity and Ca²⁺-uptake and release rates, was examined in homogenates of the superficial region of the vastus lateralis muscle from rats subjected to a single bout of treadmill running (50 m/min at 10% incline) for 2.5 min or to exhaustion. Training brought about a 12.4% increase (P < 0.05) in SR Ca²⁺-uptake rate in rested muscles. This change was not accompanied by alterations in Ca²⁺-ATPase activity, Ca²⁺-release rate, Ca²⁺ dependence of enzyme and protein contents of Ca²⁺-ATPase and ryanodine receptor. A single bout of high-intensity exercise to exhaustion evoked significant reductions (P < 0.05) in SR function, irrespective of whether or not the animals were trained. For 2.5-min run and exhausted rats, no differences existed between SR functions of untrained and trained muscles. These data suggest that high-intensity training may be capable of enhancing SR Ca²⁺-sequestering ability, and may not protect against decreasing SR function with high-intensity exercise.     

Contractile activation properties of ventricular myocardium from hypothyroid,euthyroid and juvenile rats

Contractile activation properties of intact and chemically skinned ventricular myocardium preparations were studied in juvenile (3–4 weeks old), adult euthyroid and adult hypothyroid rats. The rats were made hyperthyroid by treatment with iodine-131 and propylthiouracil. The ventricular muscle of euthyroid rats contains a mixture of isozymes of myosin while the myocardium of juvenile and hypothyroid rats are relatively pure in regard to V₁ and V₃ types of myosin respectively. No significant differences were found in either the maximum Ca²⁺ activated or rigor force developed by chemically skinned preparations in either the juvenile or hypothyroid groups compared with euthyroid adults, suggesting that there is no difference between myocardia with different isozymes of myosin in the intrinsic capacity to generate force. In the hypothyroid (V₃) preparations there was a significant shift in the force/pCa relation to the left compared with the euthyroid adult (mixture of V₁ and V₃ isozymes). The force/pCa relation for the juvenile lay in between that for the hypothyroid and euthyroid adults. The greater apparent Ca²⁺ sensitivity to activation in the hypothyroid group may relate to a slower cross-bridge cycling rate or altered Ca²⁺ kinetics in ventricular myocardium with exclusively V₃ isozyme. In intact papillary muscles differences were found in the dependence efforce on extracellular [Ca²⁺] such that a higher extracellular [Ca²⁺] was required for muscles from hypothyroid animals to attain maximum twitch force than those from juveniles. The force/frequency relations also differed, with the hypothyroid group being better able to sustain force as stimulation frequency increased than the juvenile group. Also, in the hypothyroid group, the contraction following a 3-min period of quiescence was potentiated in relation to the preceding steady-state contractions, whereas in the juvenile group it was not. These results indicate that the thyroid state may influence the pattern of calcium translocation as well as the myosin isozyme type and that both of these factors may influence contractile properties. Furthermore, the pattern of responses seen depends not only on the pattern of isozymes present but may also depend on the age of the animal because there was no simple relationship between the apparent sensitivity of the contractile apparatus for calcium and proportion of isozyme in the myocardium.     

Spatial distribution of intracellular,free Ca2+ in isolated rat parotid acini

The spatial distribution of intracellular, free Ca²⁺ ([Ca²⁺]_i) in rat parotid acini was measured by imaging fura-2 fluorescence from individual acinar cells by means of a digital imaging microscope. Upon cholinergic stimulation in a Krebs-Ringer bicarbonate buffer at (37° C), [Ca²⁺]_i increased synchronously at both the basolateral and luminal membranes as well as in all cells of the secretory endpiece, reaching peak [Ca²⁺]_i levels 1 s after stimulation. Atropine addition caused a rapid down-regulation of [Ca²⁺]_i, which, however, never reached prestimulatory levels. When acini were stimulated in a medium containing 5 nM Ca²⁺, the Ca²⁺ mobilization arising from internal pools caused an increase in [Ca²⁺]_i predominantly near the basolateral area, where the endoplasmic reticulum is located, and standing Ca²⁺ gradients were observed for up to 10 s. A mathematical model is developed to simulate the time courses of the Ca²⁺ profiles through the cytoplasm using estimated values of the Ca²⁺ diffusion coefficients and the cytosolic Ca²⁺ buffering capacity. It is concluded that under physiological conditions, the Ca²⁺ release from the endoplasmic reticulum is responsible for the activation of the basolaterally located K⁺ channels. Furthermore, Ca²⁺ influx from the interstitium is responsible for much of the rise in [Ca²⁺]_i near the luminal membranes, where the Cl^– channels are supposed to be located.