Ferret Ventricular Myocardium Research Articles

Effects of volatile anesthetics on elastic stiffness in isometrically contracting ferret ventricular myocardium.

The effects of halothane, isoflurane, and sevoflurane on elastic stiffness, which reflects the degree of cross-bridge attachment, were studied in intact cardiac muscle. Electrically stimulated (0.25 Hz, 25 degrees C), isometrically twitching right ventricular ferret papillary muscles (n = 15) at optimal length (L(max)) were subjected to sinusoidal length oscillations (40 Hz, 0.25- 0.50% of L(max) peak to peak). The amplitude and phase relationship with the resulting force oscillations was decomposed into elastic and viscous components of total stiffness in real time. Increasing extracellular Ca(2+) concentration in the presence of anesthetics to produce peak force equal to control increased elastic stiffness during relaxation, which suggests a direct effect of halothane and sevoflurane on cross bridges.

Comparison of the affinity of β‐blockers for two states of the β1‐adrenoceptor in ferret ventricular myocardium

We compared the potency of 11 clinically available beta-blockers as antagonists of the positive inotropic effects of (-)-isoprenaline and CGP12177 on ferret ventricular myocardium. (-)-CGP12177, (-)-pindolol and (-)-alprenolol were non-conventional partial agonists with intrinsic activity of 0.7, 0.2 and 0.1 respectively. All beta-blockers antagonized in a concentration-dependent and surmountable manner the positive inotropic effects of both (-)-isoprenaline and CGP12177. The potency of each beta-blocker was consistently higher against (-)-isoprenaline than against CGP12177. Two groups of beta-blockers were identified. In one group the difference between the pK(B) values of blockade against (-)-isoprenaline and CGP12177 was 1.1 - 1.6 log units ((-)-alprenolol, (-)-pindolol, (-)-bupranolol, nadolol and carvedilol). In the other group the pK(B) difference was of 2.1 - 3.0 log units ((-)-atenolol, metoprolol, bisoprolol, sotalol, (-)-propranolol and (-)-timolol). The beta-blockers competed with (-)-[(125)I]-cyanopindolol for binding to ventricular beta(1)-adrenoceptors. The binding affinities correlated with the corresponding blocking potencies against (-)-isoprenaline. On average the pK(i) values were 0.5 log units smaller than the pK(B) values against (-)-isoprenaline but 1.6 log units greater than the pK(B) values against CGP12177. In ferret ventricle the effects of (-)-isoprenaline appear to be antagonized by beta-blockers through the state of the beta(1)-adrenoceptor for which (-)-[(125)I]-cyanopindolol and beta-blockers have high affinity. The cardiostimulant effects of CGP12177 appear to be mediated through a low-affinity state of the beta(1)-adrenoceptor for which beta-blockers have low affinity.

Acetylcholine and adenosine activate the G protein-gated muscarinic K+ channel in ferret ventricular myocytes

The properties of the K+ channel activated by acetylcholine (ACh) and adenosine (Ado) were examined in single ferret ventricular myocytes using patch-clamp techniques. In the whole-cell configuration, ACh and Ado induced an inwardly rectifying K+ current and shortened the action potential duration. The effect of ACh was blocked by atropine, while the Ado effect was interrupted by 8-cyclopentyl,1,2-dipropyl xanthine, a specific Ado A1 receptor antagonist. In cell-attached recordings, ACh and Ado added to the pipette solution activated a single population of inwardly rectifying K+ channels, distinct from the iK1 channel. The channel had a slope conductance of approximately 40 pS in symmetrical 150 mM K+ solutions and a mean open time of 0.8 ms. Excision of the patch into the inside-out patch configuration in guanosine triphosphate (GTP)-free solution abolished the channel activity. The channel was reversibly reactivated by adding GTP to the intracellular side of the patch. GTP gamma S activated the channel irreversibly. When the inside-out patch was treated with the A protomer of pertussis toxin (PTX), intracellular GTP no longer activated the K+ channel. The results show that ferret ventricular myocytes possess a K+ channel activated by both muscarinic and Ado A1 receptors. Its electrophysiological properties and the gating by a PTX-sensitive G protein in a membrane-delimited fashion are identical with those of the muscarinic K+ channels in nodal and atrial tissues of other species. In conclusion, the G protein-gated muscarinic K+ channel is expressed in ferret ventricular myocardium and may underlie the direct negative inotropism of ACh and Ado in this tissue.

Mechanism of the Negative Inotropic Effect of Thiopental in Isolated Ferret Ventricular Myocardium

Thiopental's myocardial depressant effects are well known and most likely involve some alteration in intracellular Ca2+ homeostasis. The aim of this study was to investigate the mechanisms of thiopental's negative inotropic effects and its underlying mechanism in isolated ferret ventricular myocardium (which shows physiologic characteristics similar to human ventricular myocardium), and in frog ventricular myocardium, in which Ca2+ ions for myofibrillar activation are derived almost entirely from transsarcolemmal influx. The authors analyzed the effects of thiopental after beta-adrenoceptor blockade on variables of contractility and relaxation, and on the free intracellular Ca2+ transient detected with the Ca(2+)-regulated photoprotein aequorin. Thiopental's effects also were evaluated in ferret right ventricular papillary muscles in which the sarcoplasmic reticulum (SR) function was impaired by ryanodine and in frog ventricular strips with little or no SR function. At concentration > or = 10(-4) M, which is in the high range of the clinically encountered free plasma thiopental concentrations, thiopental decreased contractility and the amplitude of the intracellular Ca2+ transient. At equal peak force, peak aequorin luminescence in 10(-4) M thiopental and [Ca2+]0 > 2.25 mM was slightly smaller than that in control conditions at [Ca2+]o = 2.25 mM. This indicates that thiopental causes a small increase in myofibrillar Ca2+ sensitivity. After inactivation of sarcoplasmic reticulum Ca2+ release with 10(-6) M ryanodine, a condition in which myofibrillar activation depends almost exclusively on transsarcolemmal Ca2+ influx, thiopental caused a further decrease in contractility and in the amplitude of the intracellular Ca2+ transient, and thiopental's relative negative inotropic effect was not different from that in control muscles not exposed to ryanodine. Thiopental, > or = 10(-4) M, decreased contractility in frog ventricular myocardium. These findings indicate that the direct negative inotropic effect of thiopental results from a decrease in intracellular Ca2+ availability. At least part of thiopental's action is caused by inhibition of transsarcolemmal Ca2+ influx. These effects become apparent at concentrations routinely present during intravenous induction with thiopental.

Role of sarcoplasmic reticulum Ca 2+ release in the negative inotropic effect of ketamine HCl in isolated ferret ventricular myocardium

Role of sarcoplasmic reticulum Ca 2+ release in the negative inotropic effect of ketamine HCl in isolated ferret ventricular myocardium

Mechanism of the Negative Inotropic Effect of Propofol in Isolated Ferret Ventricular Myocardium

The aim of this study was to investigate propofol's effect on myocardial contractility and relaxation and examine its underlying mechanism of action in isolated ferret ventricular myocardium. The effects of propofol on variables of contractility and relaxation and on the free intracellular Ca++ transient detected with the Ca(++)-regulated photoprotein aequorin were analyzed. Propofol's effects were evaluated in a preparation in which the sarcoplasmic reticulum function was impaired by ryanodine. The effects of propofol's solvent, intralipid, on myocardial contractility, relaxation, and the intracellular Ca++ transient also were examined. Propofol, at concentrations of 10 microM or greater, decreased contractility and, at concentrations of 30 of microns or greater, decreased the amplitude of the intracellular Ca++ transient. At equal peak force, control peak aequorin luminescence in [Ca++]o = 2.25 mM and peak aequorin luminescence in 300 microM [Ca++]o = 2.25 mM and peak aequorin luminescence in 300 microM propofol in [Ca++]o > 2.25 mM did not differ, which suggests that propofol does not alter myofibrillar Ca++ sensitivity. After inactivation of sarcoplasmic reticulum Ca++ release with 1 microM ryanodine, a condition in which myofibrillar activation depends almost exclusively on transsarcolemmal Ca++ influx, propofol caused a decrease in contractility and in the amplitude of the intracellular Ca++ transient. Under these conditions, propofol's relative negative inotropic effect did not differ from that in control muscles not exposed to ryanodine. Propofol's solvent, 10% intralipid, exerted a modest positive inotropic effect in this preparation. The intracellular Ca++ transient was unchanged by intralipid. Neither propofol nor intralipid altered the load sensitivity of relaxation. These findings suggest that the negative inotropic effect of propofol results from a decrease in intracellular Ca++ availability with no changes in myofibrillar Ca++ sensitivity. At least part of propofol's action is attributable to inhibition of transsarcolemmal Ca++ influx.

Mechanism of the direct, negative inotropic effect of etomidate in isolated ferret ventricular myocardium.

Etomidate exerts a mild, positive inotropic effect in rat ventricular myocardium, yet has a negative inotropic effect in isolated rabbit ventricular myocardium. The aim of this study was to investigate the mechanisms of etomidate's inotropic effect and its underlying mechanism in isolated ferret ventricular myocardium (which shows similar physiologic characteristics as human ventricular myocardium) and in frog ventricular myocardium, in which Ca++ ions for myofibrillar activation are derived almost entirely from transsarcolemmal influx. The authors analyzed the effects of etomidate after beta-adrenoceptor blockade on variables of contractility and relaxation, and on the free intracellular Ca++ transient detected with the Ca+(+)-regulated photoprotein aequorin. Etomidate's effects were also evaluated in ferret right ventricular papillary muscles in which the sarcoplasmic reticulum (SR) function was impaired by ryanodine, and in frog ventricular strips with little or no SR function. At concentrations > or = 3 micrograms/ml, which by far exceed the clinically useful concentration range, etomidate decreased contractility and the amplitude of the intracellular Ca++ transient. At equal peak force, control peak aequorin luminescence in [Ca++]o = 2.25 mM and peak aequorin luminescence in etomidate 10 micrograms/ml and [Ca++]o > 2.25 mM did not differ, which indicates that etomidate does not alter myofibrillar Ca++ sensitivity. After inactivation of sarcoplasmic reticulum Ca++ release with ryanodine 10(-6) M, a condition in which myofibrillar activation depends almost exclusively on transsarcolemmal Ca++ influx, etomidate caused a decrease in contractility and in the amplitude of the intracellular Ca++ transient, and etomidate's relative negative inotropic effect was not different from that in control muscles not exposed to ryanodine. Etomidate 10 micrograms/ml decreased contractility in frog ventricular myocardium. These findings indicate that the direct negative inotropic effect of etomidate results from a decrease in intracellular Ca++ availability with no changes in myofibrillar Ca++ sensitivity. At least part of etomidate's action is caused by inhibition of transsarcolemmal Ca++ influx. Yet, these effects become apparent only at concentrations that are at least one order of magnitude larger than those encountered in clinical practice.

Modulation of basal L‐type Ca2+ current by adenosine in ferret isolated right ventricular myocytes.

1. The whole-cell configuration of the gigaohm seal voltage clamp and an internal perfusion technique were used to study the effects of adenosine on the basal L-type Ca2+ current (ICa) in enzymatically isolated right ventricular myocytes of ferrets. Basal L-type ICa was isolated by using a Na(+)- and K(+)-free saline (replacement by N-methyl-D-glucamine+, Cs+ and TEA+, respectively). All experiments were conducted at room temperature (22-24 degrees C). 2. Basal ICa was markedly reduced during exposure to adenosine in a concentration-dependent manner with a half-inhibitory concentration (IC50) of 0.3 microM and maximum inhibition of 35%. This effect was completely abolished by 50 nM 8-cyclopentyl-1,3-dipropylxanthine (CPDPX), a specific A1 adenosine receptor antagonist with an inhibition constant, Ki = 0.48 nM. Inhibition was also observed in the presence of 1 microM atropine. 3. Adenosine decreased basal ICa by decreasing the peak amplitude of ICa without significantly altering (i) the voltage dependence of the current-voltage relationship, (ii) the apparent reversal potential, (iii) the voltage dependence of steady-state activation and inactivation, (iv) the kinetics of inactivation at 0 mV, and (v) the kinetics of recovery from inactivation at -70 mV. 4. Pretreatment of cells with 0.4 microns/ml pertussis toxin (PTX) for 4 h at 37 degrees C produced greater than 90% ADP ribosylation of PTX-sensitive G proteins. PTX pretreatment significantly attenuated the adenosine-mediated decrease in ICa (35% in control; 4.6% after PTX pretreatment). 5. The peptide inhibitor (PKI) of cyclic AMP-dependent protein kinase A at a concentration of 2 microM neither inhibited basal ICa nor attenuated the effects of adenosine on basal ICa. However, PKI decreased the stimulatory effects of 100 microM cAMP on ICa. 6. Increasing intracellular cAMP to a supra-saturable level by using 10 mM cAMP and 100 microM papaverine did not prevent adenosine from inhibiting ICa. 7. Consistent with the reduction of basal ICa, adenosine produced an inhibitory effect on the action potential under basal conditions, i.e. hyperpolarization of the plateau phase and marked shortening of action potential duration. These effects were concentration dependent. 8. These results demonstrate a reduction of the basal L-type ICa by adenosine in ferret ventricular myocardium. This reduction is not mediated by modification of voltage-dependent properties of macroscopic ICa. The shortening of action potentials may be explained in part by the reduction in ICa.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanism of the direct, negative inotropic effect of ketamine in isolated ferret and frog ventricular myocardium.

Ketamine exerts both an indirect, positive inotropic effect and a direct, negative inotropic effect in isolated ferret ventricular myocardium. This negative inotropic effect becomes apparent after inactivation of the sympathetic neuroeffector junction. The aim of this study was to investigate the mechanisms of ketamine's intrinsic negative inotropic effect. The authors analyzed the effects of ketamine after beta-adrenoceptor blockade on variables of contractility and relaxation, and on the free intracellular Ca++ transient detected with the Ca(++)-regulated photoprotein aequorin. Ketamine's effects were also evaluated in a preparation in which the sarcoplasmic reticulum (SR) function was impaired by ryanodine, and in frog ventricular myocardium in which the SR is poorly developed. Ketamine at concentrations > or = 3.3 x 10(-5) M decreased contractility and the amplitude of the intracellular Ca++ transient. After inactivation of sarcoplasmic reticulum Ca++ release with 10(-6) M ryanodine, a condition in which myofibrillar activation depends almost exclusively on transsarcolemmal Ca++ influx, ketamine caused a decrease in contractility and in the amplitude of the intracellular Ca++ transient, and ketamine's relative negative inotropic effect was not different from that in control muscles not exposed to ryanodine. Furthermore, > or = 10(-4) M ketamine decreased contractility in frog ventricular myocardium, a species that is almost entirely dependent on transsarcolemmal Ca++ influx for its myofibrillar activation. These findings indicate that the direct negative inotropic effect of ketamine results from a decrease in intracellular Ca++ availability with no changes in myofibrillar Ca++ sensitivity. At least part of ketamine's action is caused by inhibition of transsarcolemmal Ca++ influx.

Effects of extracellular ATP on ICa, [Ca2+]i, and contraction in isolated ferret ventricular myocytes

The effects of extracellular ATP on the voltage-activated "L-type" Ca current (ICa), action potential, resting and transient intracellular Ca2+ levels, and cell contraction were examined in enzymatically isolated myocytes from the right ventricles of ferrets. With the use of the whole cell patch-clamp technique, extracellular ATP (10(-7) to 10(-3) M) inhibited ICa in a time- and concentration-dependent manner. ATP decreased the peak amplitude of ICa without altering the residual current at the end of 500-ms clamp steps. The concentration-response relationship for ATP inhibition of ICa was well described by a conventional Michaelis-Menten relationship with a half-maximal inhibitory concentration of 1 microM and a maximal effect of 50%. Consistent with its inhibitory effect on ICa, ATP hyperpolarized the plateau phase and shortened the action potential duration. In fura-2-loaded myocytes, extracellular ATP did not change the resting myoplasmic Ca2+ levels; however, when current was elicited under voltage-clamp conditions, ATP both decreased the myoplasmic intracellular Ca2+ transient and inhibited the degree of cell shortening. Our results suggest that ATP could be a genuine and potent extracellular modulator of cardiac function in ferret ventricular myocardium.

Cellular mechanisms of paired electrical stimulation in ferret ventricular myocardium: relationship between myocardial force and stimulus interval change.

The subcellular mechanisms of twitch-force potentiation with paired electrical stimulation was studied in ferret ventricular myocardium using the bioluminescent calcium indicator aequorin. It is demonstrated for the first time that interpolation of an extrasystole in a train of conditioned twitches results in a beat-to-beat change in [Ca2+]i and force. Steady-state twitch force and Ca2+i were increased with paired stimulation. Increased [Ca2+]o in the setting of paired stimulation resulted in an increase in the amplitude of the postextrasystole and associated Ca2+ transient. Verapamil, a Ca2+ channel antagonist, had the opposite effect of increased [Ca2+]o. Postextrasystole potentiation was still present, but diminished in amplitude. These results indicate that postextrasystole potentiation is in part due to a verapamil-depletable store (Ca2+). Postextrasystole potentiation is therefore predominantly dependent on sarcoplasmic reticulum (SR) Ca2+ loading. Ryanodine, an alkaloid which induces Ca2+ leakage from the SR, abolished postextrasystole potentiation; however, in the presence of ryanodine the extrasystole was potentiated. Caffeine, a phosphodiesterase inhibitor which induces SR Ca2+ release and impairs uptake, also abolished postextrasystole potentiation. As with ryanodine there was resultant potentiation of the extrasystole. In the case of caffeine the calcium transient consisted of a second slow component associated with extrasystole twitch potentiation. The results are consistent with sarcolemmal Ca2+ influx playing a role in potentiation of the extrasystole in the presence of an impaired SR. These data indicate that transsarcolemmal Ca2+ influx in the presence of impaired intracellular Ca2+ buffering can directly activate the myofilaments in agreement with reports on human myocardium.

The effects of cocaine on intracellular Ca2+ handling and myofilament Ca2+ responsiveness of ferret ventricular myocardium

1. When ferret right ventricular papillary muscles were stimulated with threshold punctate pulses (0.33 Hz; 30 degrees C), cocaine, 10(-5) M, increased peak tension development from 815 +/- 120 to 1125 +/- 180 mg (P less than 0.05) and increased the rate of relaxation from peak tension (time to 80% decline from peak tension decreased from 155 +/- 11 to 144 +/- 11 ms; P less than 0.05). These changes in the twitch were associated with comparable changes in the amplitude and time course of the calcium transient measured with aequorin (amplitude increased from 62 +/- 4 to 90 +/- 7% (P less than 0.05) of maximal values; time to 80% decline from peak amplitude decreased from 84 +/- 8 to 64 +/- 3 ms; P less than 0.05). These effects were markedly attenuated in the presence of the beta-adrenoceptor-blocking agent, propranolol, 6 x 10(-7) M, or by maximization of catecholamine release from the adrenergic nerve endings with field pulses of suprathreshold strength, indicating that catecholamine release from the adrenergic nerve endings is responsible for the positive inotropic and lusitropic responses to low and moderate doses of cocaine (i.e., less than or equal to 10(-5) M). 2. High doses of cocaine (i.e., greater than 10(-5) M) produced negative inotropic and lusitropic effects that were associated with a decreased amplitude and prolonged duration of the calcium transient. 3. In aequorin-loaded intact fibres, cocaine 10(-5) M did not affect the force-calcium relationship unless catecholamines were present. Cocaine, 10(-5) M, significantly shifted the force-calcium relationship of saponin-skinned muscles (pCa50 = 6.14 +/- 0.05 versus 5.92 +/- 0.07; P less than 0.05), indicating reduced responsiveness of the myofilaments to calcium. F. (maximal Ca2+-activated force) was reduced to 58% of control in the presence of 10- M cocaine, while the slope of the calcium-force curve remained unchanged. These data indicate that cocaine may also decrease myofilament calcium sensitivity and maximal calciumactivated force, via mechanisms independent of catecholamines, when cellular diffusion barriers are eliminated.

Negative inotropy of halogenated anesthetics in ferret ventricular myocardium

The effects of the volatile anesthetics halothane, enflurane, and isoflurane on amplitude and time variables of contraction and relaxation in isometric, isotonic, and zero-load clamped twitches of isolated right ventricular ferret papillary muscles (n = 24) were compared with those of changing extracellular Ca2+ concentration [( Ca2+]o). Contraction and relaxation variables were compared at equal contraction amplitudes to determine whether the studied anesthetics exert specific effects on contraction and relaxation other than those that may exist as a consequence of their negative inotropic effect. Both changes in [Ca2+]o and anesthetic concentrations had quantitatively and qualitatively similar effects on the following variables of contraction amplitude: peak isometric developed force (DF), maximal extent of shortening (DL), and maximal unloaded velocity of shortening (MUVS). Analysis of anesthetic concentration-effect curves normalized for effects to changes in [Ca2+]o demonstrated that anesthetic effects relative to those of changes in [Ca2+]o were greater on DF greater than DL greater than MUVS. When compared with twitches of equal amplitude in low [Ca2+]o, halothane, enflurane, and isoflurane accelerated isometric and isotonic relaxation. These data are consistent with the hypothesis that the negative inotropic effects of halothane, enflurane, and isoflurane are mostly a consequence of a reduction of intracellular Ca2+ availability and that anesthetic-induced decreases in myofibrillar Ca2+ responsiveness play only a minor role.

Pharmacological characteristics of adenosine-induced inhibitory action in ferret ventricular myocardium

Pharmacological characteristics of adenosine-induced inhibitory action in ferret ventricular myocardium

S-33 - Negative inotropic effect mediated by adenosine receptors in the ferret ventricular myocardium

S-33 - Negative inotropic effect mediated by adenosine receptors in the ferret ventricular myocardium

Preponderance of ?- over ?-adrenoceptors in mediating the positive inotropic effect of phenylephrine in the ferret ventricular myocardium

[3H]prazosin bound to the membrane fraction derived from the ferret ventricular muscle with high affinity in a saturable manner (Kd = 0.25 nmol/l and Bmax = 27 fmol/mg protein in the right ventricle). [3H]CGP-12177, a beta-adrenoceptor ligand, bound to the membrane fraction with a Kd value of 0.29 nmol/l and a Bmax of 42 fmol/mg protein. In the isolated ferret papillary muscle driven at 1 Hz at 37 degrees C, phenylephrine elicited a concentration-dependent positive inotropic effect. The maximal effect of phenylephrine was comparable to that of isoprenaline. Prazosin (0.3 mumol/l) shifted the concentration-response curve for phenylephrine slightly but significantly to the right, the maximal response being unaffected. In contrast, bupranolol (0.3 mumol/l) shifted the curve for phenylephrine markedly downwards: the maximal response was depressed significantly to 40% and the curve became less steep. In the presence of prazosin and bupranolol the curve was shifted to the right, being essentially parallel to the control curve. These results indicate that in the ferret ventricular myocardium both alpha- and beta-adrenoceptors mediate the positive inotropic effect of phenylephrine. The extent of contribution of the two classes of adrenoceptor is quite different from that in other mammalian species. In the ferret heart, beta-adrenoceptors predominate over alpha-adrenoceptors in mediating the positive inotropic effect of phenylephrine, although the number of beta-adrenoceptors is not especially high when compared with other species.

The effects of cyanide on intracellular ionic exchange in ferret and rat ventricular myocardium

The effects of cyanide on Ca2+ exchange in isolated ventricular myocytes and on the intracellular concentrations of Ca2+, Na+ and H+ have been investigated to assess the contribution that mitochondria might play in cellular Ca2+ metabolism. Ionic levels were measured with ion-selective electrodes. KCN (2.5 mM) inhibited a component of Ca2+ exchange in myocytes that could be attributed to mitochondrial exchange, but was without effect on non-mitochondrial Ca2+ exchange. NaCN (2.5 mM) caused a transient reduction of [H+]i, [Na+]i and [Ca2+]i when applied to the superfusate bathing ventricular trabeculae or papillary muscles. The transient changes of [Na+]i were accentuated when the preparation was exposed to a solution which would be expected to increase the cellular calcium content. The reduction of [Na+]i which accompanies a reduction of the extracellular sodium concentration, [Na]o, was attenuated in the presence of NaCN, but the intracellular acidosis resulting from a reduction of [Na]o was unaffected by NaCN. A small, but significant, rise of [Ca2+]i accompanied a reduction of [Na]o but only when NaCN was present in the superfusate. It is concluded that cyanide ions have a reasonably specific action on cardiac cellular ionic metabolism. Its primary action is to prevent mitochondrial Ca2+ sequestration. It is postulated that a Na+/H+ exchange, possibly at the sarcolemma, could account for some of the changes to sarcoplasmic ionic levels observed. In a solution of low [Na]o, it is concluded that mitochondria could sequester at least 30% of the calcium accumulated by the cell even though the sarcoplasmic [Ca2+] does not exceed 0.3 microM.

Intracellular calcium transients underlying the short‐term force‐interval relationship in ferret ventricular myocardium.

The influence of short-term changes in stimulation pattern, both on the strength of contraction and on the amplitude of intracellular free-Ca2+ transients, was investigated in ferret papillary muscles. Intracellular free-Ca2+ concentration ([Ca2+]) was assessed from the luminescence emitted from muscles microinjected with the Ca2+-sensitive photoprotein aequorin. The relationships between the strength of contraction and changes in stimulation pattern lasting 1-2 beats could be described by monoexponential functions, all with very similar time constants (approximately 750 ms at 30 degrees C). Over the entire range that could be obtained, the strength of contraction, quantified by either peak tension or peak rate of tension development, was found to be linearly correlated with peak estimated [Ca2+]. Potential errors in the estimation of [Ca2+] from aequorin luminescence were analysed. To assess the influence of spatial non-homogeneities of [Ca2+] on the estimate of [Ca2+], a model of Ca2+ diffusion in heart muscle was developed. The possible effect of using an inaccurate calibration curve was also examined. The results of these analyses indicate that [Ca2+] estimated from aequorin luminescence should be proportional to, if not equal to, true spatial average [Ca2+] (errors less than 7%). Given the conclusion of the analysis described above, it is inferred from points 2 and 3 that the relationships between peak spatial average [Ca2+] and short-term changes in stimulation pattern are also represented by monoexponential functions, with time constants closely similar to those for the mechanical measurements. Exposure to ryanodine, a substance believed to inhibit the release of Ca2+ from sarcoplasmic reticulum, produced striking alterations in the pattern of variations in [Ca2+] mentioned above. These alterations are consistent with the hypothesis that the functions described above depend essentially upon properties of the sarcoplasmic reticulum.

Neutral carrier ion-selective microelectrodes for measurement of intracellular free calcium

This paper describes the making and testing of calcium-selective microelectrodes for measurement of intracellular [free Ca 2+] levels. Pipettes of tip outer diameter down to 0.4 μm were siliconized by a novel and easy method of vapor treatment. The tips were filled with a sensor mixture using the neutral ligand and solvent of Oehme et al. (Oehme, M., Kessler, M. and Simon, W. (1976) Chimia (Aarau) 30, 204–206) but with very hydrophobic cations replacing Na + in the salt component. This change improved electrode stability and greatly reduced hysteresis in the responses to changing [Ca 2+] levels. Lowering the Ca 2+ concentration in the internal electrolyte also increased electrode lifetime. Electrodes showed a Nernstian response to [Ca 2+] down to 1 μM free concentration in 0.1 M KCl, and usually a useful response to below 100 nM Ca 2+. Selectivity for Ca 2+ over Mg 2+ and H + was sufficiently high that typical free intracellular levels of Mg 2+ and H + caused negligible interference. Selectivity for Ca 2+ over Na + was adequate for cells with 10 −2 M free Na +, but higher levels could raise significantly the detection limit for Ca 2+. Preliminary measurements of [free Ca 2+] have been made in frog skeletal muscle, ferret ventricular myocardium, and early embryos of Xenopus laevis. Relative merits of Ca 2+ microelectrodes and optical indicators are discussed.