Different inhibition patterns of tedisamil for fast and slowly inactivating transient outward current in rat ventricular myocytes

Experimental evidence increasingly implicates the beta-amyloid peptide in the pathogenesis of Alzheimer's disease. Beta-amyloid filaments dramatically accumulate in the neuritic plaques and vascular deposits as the result of the brain's inability to clear these structures. In this paper, we demonstrate that in addition to the intrinsic stability of A beta N-42, the time dependent generation of irreversibly associated A beta dimers and tetramers incorporated into A beta filaments are themselves resistant to proteolytic degradation. The presence of post-translational modifications such as isomerization of aspartyls 1 and 7, cyclization of glutamyl 3 to pyroglutamyl and oxidation of methionyl 35, further contribute to the insolubility and stability of A beta. All these factors promote the accumulation of neurotoxic amyloid in the brains of patients with Alzheimer's disease, and should be considered in therapeutic strategies directed towards the dissociation of the brain's A beta filaments.     

Anoxia decreases the transient K+ outward current in isolated ventricular heart cells of the mouse

Transient K+ outward currents (Ito) were measured in enzymatically isolated ventricular mouse heart cells with a patch clamp technique in the whole cell configuration. Exposure of the cells to substrate-free anoxia gradually decreased both the peak and the late Ito. The inactivation time course of Ito was fitted with two exponentials. After 4-10 min of anoxia, the contribution of the fast and slow exponential decreased to 60 +/- 7% and 62 +/- 4% of the control value and recovered after reoxygenation within 1-3 min to 84 +/- 5% and 75 +/- 6% (n = 10; all mean +/- SEM), respectively. The time constants of the exponentials were invariant to anoxia. Voltage dependence of activation and inactivation of Ito were not influenced by anoxia. Application of stimulators of protein kinase A and C, cGMP- dependent protein kinase, or of the oxidant diamide during anoxia did not recover Ito. It is concluded that under conditions of metabolic stress, Ito is reversibly down-regulated leaving inactivation kinetics unchanged. The underlying mechanism is as yet unknown but does neither involve a decreased activity of protein kinase A, protein kinase C, nor c-GMP dependent protein kinase.     

Lamotrigine may limit pathological excitation in the hippocampus by modulating a transient potassium outward current

Actions of the new antiepileptic drug lamotrigine were characterised using whole cell patch clamp recordings from rat CA1 pyramidal cells in vitro. The results suggest that lamotrigine, besides its previously described effect on the fast sodium inward current and calcium currents, modulates the transient potassium outward current ID. This may be an effective mechanism to inhibit pathological excitation.     

Characteristics of a fast transient outward current in guinea pig trigeminal motoneurons

A fast transient voltage dependent outward current (TOC) in trigeminal motoneurons (TMNs) was studied in guinea pig brainstem slices by use of sharp electrodes in combination with single electrode voltage clamp techniques. In solutions containing TTX, low Ca2+/Mn2+ and 20 mM TEA this current activated around -55 to -60 mV from holding potentials negative to resting potential, obtained its peak amplitude within 5 ms and decayed as a single exponential with a time constant of 6-8 ms. Half maximal values for inactivation and activation were -72 and -37 mV, respectively. Bath application of 5 mM 4-AP suppressed this current by approximately 90% and eliminated the early depolarizing transient membrane rectification observed in response to a constant depolarizing current pulse, prolonged the action potential duration, and reduced the threshold voltage and delay to onset of the action potential. It is suggested that this current resembles the typical A-current observed in many CNS neurons and, as a result of its voltage and time dependent properties, could contribute to control of motoneuronal discharge and timing of burst onset during rhythmical jaw movements. Therefore, any cellular models of masticatory activity should include the properties of this current.     

The calcium-independent transient outward potassium current in isolated ferret right ventricular myocytes. I. Basic characterization and kinetic analysis

Enzymatically isolated myocytes from ferret right ventricles (12-16 wk, male) were studied using the whole cell patch clamp technique. The macroscopic properties of a transient outward K+ current I(to) were quantified. I(to) is selective for K+, with a PNa/PK of 0.082. Activation of I(to) is a voltage-dependent process, with both activation and inactivation being independent of Na+ or Ca2+ influx. Steady-state inactivation is well described by a single Boltzmann relationship (V1/2 = -13.5 mV; k = 5.6 mV). Substantial inactivation can occur during a subthreshold depolarization without any measurable macroscopic current. Both development of and recovery from inactivation are well described by single exponential processes. Ensemble averages of single I(to) channel currents recorded in cell-attached patches reproduce macroscopic I(to) and indicate that inactivation is complete at depolarized potentials. The overall inactivation/recovery time constant curve has a bell-shaped potential dependence that peaks between -10 and -20 mV, with time constants (22 degrees C) ranging from 23 ms (-90 mV) to 304 ms (-10 mV). Steady-state activation displays a sigmoidal dependence on membrane potential, with a net aggregate half-activation potential of +22.5 mV. Activation kinetics (0 to +70 mV, 22 degrees C) are rapid, with I(to) peaking in approximately 5-15 ms at +50 mV. Experiments conducted at reduced temperatures (12 degrees C) demonstrate that activation occurs with a time delay. A nonlinear least-squares analysis indicates that three closed kinetic states are necessary and sufficient to model activation. Derived time constants of activation (22 degrees C) ranged from 10 ms (+10 mV) to 2 ms (+70 mV). Within the framework of Hodgkin-Huxley formalism, Ito gating can be described using an a3i formulation.     

Inhibition by taurine of the inwardly rectifying K+ current in guinea pig ventricular cardiomyocytes

Effects of taurine on the inwardly rectifying K+ current (IK1) in isolated guinea pig ventricular cardiomyocytes were examined using patch voltage-clamp methods. All experiments were performed at 36 degrees C. Taurine (10-20 mM) increased the action potential duration, but failed to affect the resting potential. Holding potential was maintained at -30 mV. The current was activated with an inwardly going rectification, and was completely blocked by Ba2+ (2 mM). Taurine inhibited IK1 at - 120 mV by 28.3+/-1.1% (n=6, P < 0.05) at 10 mM and by 36.0+/-2.1% (n=6, P < 0.01) at 20 mM. The reversal potential was shifted in the hyperpolarizing direction by 3.7+/-0.6 mV (n=6) at 20 mM. In inside-out patch-clamp experiments, the amplitude of unitary channels was -2.7+/-0.3 pA (n=21) at -90 mV. Symmetrical high-K+ (150 mM) solutions in both bath and pipette were used. The channel conductance was 32+/-2 pS (n=9). Taurine did not affect channel conductance, but markedly decreased the open probability at - 120 mV of channel by 21.5+/-2.4% (n=8, P < 0.01) at 10 mM, and by 56.7+/-3.8% (n=8, P < 0.001) at 20 mM. These responses were almost reversible. These results suggest that taurine directly modulates the open probability of the inwardly rectifying K+ current, resulting in regulation of the functions of heart cells.     

Characterization of a slowly inactivating outward current in adult mouse ventricular myocytes

We recently have reported that suppression of the slowly inactivating component of the outward current, Islow, in ventricular myocytes of transgenic mice (long QT mice) overexpressing the N-terminal fragment and S1 segment of Kv1.1 resulted in a significant prolongation of action potential duration and the QT interval. Here we describe the detailed biophysical properties and physiological role of Islow by applying the whole-cell patch-clamp technique at both room temperature and 37 degreesC. This current activates rapidly with time constants ranging from 3.8+/-0.8 ms at -20 mV to 2.1+/-0.5 ms at 50 mV at room temperature. The half-activation voltage and slope factor are -12.5+/-2.6 mV and 7. 7+/-1.0 mV, respectively. The inactivation of this current is slow compared with the fast inactivating component Ito, with time constants of approximately 100 ms at 37 degreesC. The steady-state inactivation of Islow is not temperature-dependent, with half-inactivation voltages and slope factors of -35.1+/-1.3 and -5. 4+/-0.4 mV at 37 degreesC, and -37.6+/-1.8 and -5.8+/-0.6 mV at room temperature. Double exponentials were required to describe the time-dependent recovery of Islow from steady-state inactivation, with time constants of 233+/-34 and 3730+/-702 ms at 37 degreesC, and 830+/-240 and 8680+/-2410 ms at room temperature. Islow is highly sensitive to 4-aminopyridine but is insensitive to tetraethylammonium, alpha-dendrotoxin, and E-4031. Stimulation with action-potential waveforms under voltage-clamp mode revealed that this current plays an important role in the early and middle phases of repolarization of the cardiac action potential. We conclude that the biophysical properties and pharmacological profiles of Islow are similar to those of Kv1.5-encoded currents.     

A transient outward cationic current activated by Na+ influx into frog visual neurones in vitro

Location peculiarities of insular neurones implicated in the regulation of gastrointestinal motility have been studied in acute experiments on rats. After microinjection of a horseradish peroxidase solution in a part of the dorsal vagal complex that receive gastric afferent inputs, retrogradely labelled cell bodies are observed in a certain area of the agranular and disgranular insular cortex. Electrical stimulation of the insular cortex area had no significant effect on heart and respiration rate but had evoked gastric tone changes. These results suggest that the insular cortex contains a specific cell group that provides direct output to the bulbar 'gastric' centre and takes part in regulation of gastrointestinal functions.     

Novel, ultraslow inactivating sodium current in human ventricular cardiomyocytes

BACKGROUND: Alterations in K+ channel expression and gating are thought to be the major cause of action potential remodeling in heart failure (HF). We previously reported the existence of a late Na+ current (INaL) in cardiomyocytes of dogs with chronic HF, which suggested the importance of the Na+ channel in this remodeling process. The present study examined whether this INaL exists in cardiomyocytes isolated from normal and failing human hearts. METHODS AND RESULTS: A whole-cell patch-clamp technique was used to measure ion currents in cardiomyocytes isolated from the left ventricle of explanted hearts from 10 patients with end-stage HF and from 3 normal hearts. We found INaL was activated at a membrane potential of -60 mV with maximum density (0.34+/-0.05 pA/pF) at -30 mV in cardiomyocytes of both normal and failing hearts. The steady-state availability was sigmoidal, with an averaged midpoint potential of -94+/-2 mV and a slope factor of 6.9+/-0.1 mV. The current was reversibly blocked by the Na+ channel blockers tetrodotoxin (IC50=1.5 micromol/L) and saxitoxin (IC50=98 nmol/L) in a dose-dependent manner. Both inactivation and reactivation of INaL had an ultraslow time course (tau approximately 0.6 seconds) and were independent of voltage. The amplitude of INaL was independent of the peak transient Na+ current. CONCLUSIONS: Cardiomyocytes isolated from normal and explanted failing human hearts express INaL characterized by an ultraslow voltage-independent inactivation and reactivation.     

Prostaglandins suppress an outward potassium current in embryonic rat sensory neurons

One of the challenges in understanding ciliary and flagellar motility is determining the mechanisms that locally regulate dynein-driven microtubule sliding. Our recent studies demonstrated that microtubule sliding, in Chlamydomonas flagella, is regulated by phosphorylation. However, the regulatory proteins remain unknown. Here we identify the 138-kD intermediate chain of inner arm dynein I1 as the critical phosphoprotein required for regulation of motility. This conclusion is founded on the results of three different experimental approaches. First, genetic analysis and functional assays revealed that regulation of microtubule sliding, by phosphorylation, requires inner arm dynein I1. Second, in vitro phosphorylation indicated the 138-kD intermediate chain of I1 is the only phosphorylated subunit. Third, in vitro reconstitution demonstrated that phosphorylation and dephosphorylation of the 138-kD intermediate chain inhibits and restores wild-type microtubule sliding, respectively. We conclude that change in phosphorylation of the 138-kD intermediate chain of I1 regulates dynein-driven microtubule sliding. Moreover, based on these and other data, we predict that regulation of I1 activity is involved in modulation of flagellar waveform.     

Functional knockout of the transient outward current, long-QT syndrome, and cardiac remodeling in mice expressing a dominant-negative Kv4 alpha subunit

Amino acid changes S180A (S-->A at site 180), H197Y, Y277F, T285A, and A308S are known to shift the maximum wavelength of absorption (lambda max) of red and green visual pigments toward blue, essentially in an additive fashion. To test the generality of this "five-sites" rule, we have determined the partial amino acid sequences of red and green pigments from five mammalian orders (Artiodactyla, Carnivora, Lagomorpha, Perissodactyla, and Rodentia). The result suggests that cat (Felis catus), dog (Canis familiaris), and goat (Capra hircus) pigments all with AHYTA at the five critical sites have lambda max values of approximately 530 nm, whereas rat (Rattus norvegicus) pigment with AYYTS has a lambda max value of approximately 510 nm, which is accurately predicted by the five-sites rule. However, the observed lambda max values of the orthologous pigments of European rabbit (Oryctolagus cuniculus), white-tailed deer (Odocoileus virginianus), gray squirrel (Sciurus carolinensis), and guinea pig (Cavia procellus) are consistently more than 10 nm higher than the predicted values, suggesting the existence of additional molecular mechanisms for red and green color vision. The inferred amino acid sequences of ancestral organisms suggest that the extant mammalian red and green pigments appear to have evolved from a single ancestral green-red hybrid pigment by directed amino acid substitutions.     

Prostaglandin E2 and 4-aminopyridine prevent the lipopolysaccharide-induced outwardly rectifying potassium current and interleukin-1beta production in cultured rat microglia

Brain inflammation includes microglial activation and enhanced production of diffusible chemical mediators, including prostaglandin E2. Prostaglandin E2 is generally considered a proinflammatory molecule, but it also promotes neuronal survival and down-regulates some aspects of microglial activation. It remains unknown, however, if and how prostaglandin E2 prevents microglial activation. In primary culture, microglial activation is predicted by a characteristic pattern of whole-cell potassium currents and interleukin-1beta production. We investigated if prostaglandin E2 could alter these currents and, if so, whether these currents are necessary for microglial activation. Microglia were isolated from mixed cell cultures prepared from neonatal rat brains and exposed to 0-10 microM prostaglandin E2 and lipopolysaccharide for 24 h. Currents were elicited by using standard patch-clamp technique, and interleukin-1beta production was measured by ELISA. Peak outward current densities in microglia treated with lipopolysaccharide plus prostaglandin E2 (10 nM) were reduced significantly from those of cells treated with lipopolysaccharide alone. Prostaglandin E2 and 4-aminopyridine (a blocker of outward potassium currents) also significantly reduced interleukin-1beta production. Thus, although prostaglandin E2 is classified generally as a proinflammatory chemical, it has complex roles in brain inflammation that include preventing microglial activation, perhaps by reducing the outward potassium current.     

Actions of taurine on the L-type Ca2+ channel current in guinea pig ventricular cardiomyocytes

This article presents a new method for analyzing the spreading of skin erythemas. These occur as a result of the cutaneous vascular axon reflex which can be evoked by a noxious stimulation of the skin. Series of true-color images of the observed skin patch were recorded using a video camera. The images were digitized and stored on computer disk. The delineation of the reddening was segmented for every image of the sequence by a newly developed image processing method. Each image taken after the noxious stimulation was compared with the baseline before the stimulation and each image point was classified as: "unchanged" or "changed skin color." To improve the classification the CIE L*a*b* color space was used. The boundaries of the erythema were extracted from the resulting binary images. Every image of a sequence was analyzed in the same way in order to follow the time course of the flare response. The erythema reaction could be determined in an objective way using this methods. The automatically detected flare sizes were independent of human observers and had a high spatial and temporal resolution. It was used for a crossover study to assess the power of new drugs which modify the blood flow of the skin induced by an intradermal histamine application.     

L- and T-type calcium currents differ in finch and rat ventricular cardiomyocytes

We compared L-type Ca current (ICaL) and T-type Ca current (ICaT) in finch and rat myocytes, using whole-cell patch clamp techniques. Cell capacitance averaged 50 +/- 4 pF in finch (n = 25) v 145 +/- 8 pF in rat (n = 38) cells, P < 0.001. In cells bathed in 1 mM Cao at 22 degrees C, peak ICaL amplitude, during a voltage clamp step (10 mM EGTA in pipette) from -45 mV to -5 mV, averaged 10.5 +/- 0.3 pA/pF in finch v 6.9 +/- 0.6 pA/pF, P < 0.001 in rat cells. ICaL inactivation kinetics were faster in finch (4.6 +/- 0.3 ms) than in rat (13.4 +/- 1.3 ms) cells. P < 0.001. ICaT was not detectable in rat cells (2 mM bathing [Ca]); but in finch cells, a large ICaT which averaged 6.8 +/- 1.4 pA/pF was activated at -30 mV and was relatively insensitive to nitrendipine (0.1 microM). The distinctive features of ICaL and ICaT in finch cells may have a role in the ability of the finch to achieve a very rapid heart rate. They may also facilitate excitation-Ca2+ release coupling in finch ventricular cells which are devoid of T tubules and have relatively few junctions between the sarcolemma and the sarcoplasmic reticulum.     

Purinergic facilitation of ATP-sensitive potassium current in rat ventricular myocytes

Patients who cannot be reperfused after thrombolytic therapy have a high mortality rate. Noninvasive clinical markers of reperfusion have been widely studied, yet their prognostic significance remains unclear. To assess the prognostic value of commonly used noninvasive clinical markers of early reperfusion we studied 327 patients who received intravenous thrombolytic treatment (1.5 MU streptokinase in 1 hour or 100 mg alteplase in 3 hours) within 6 hours of acute infarction. Successful clinical reperfusion (SCR) was defined as the presence of at least two of the following criteria at 2 hours after thrombolytic treatment: (1) significant relief of pain (a 5-point reduction on a 1 to 10 subjective scale), (2) > or =50% reduction of sum of ST segment elevation, and (3) abrupt initial increase of creatine kinase levels (more than twofold over the upper-normal or baseline elevated values). Clinical variables that were significantly associated by univariate analysis were tested by multivariate analysis to obtain independent predictors of 30-day mortality rate. SCR was present in 210 (64%) patients (group 1), and absent in 117 (36%) patients (group 2). The groups were similar for most baseline characteristics, although group 2 patients were slightly older (mean 60 vs 57 years, p < 0.02). Thirty-day outcomes for group 2 patients compared with group 1 patients were heart failure in 23.1% and 10.5% (p < 0.005), progression to cardiogenic shock in 12.8% and 0.5%, (p < 0.00001), and death in 16.2% and 3.8% (p < 0.0001), respectively. By multivariate analysis the Killip class at admission (p < 0.00001), the absence of SCR (p = 0.017), anterior infarct location (p = 0.021), and age (p = 0.03) were independent predictors of mortality rate, and sex (p = 0.051) had borderline significance. The absence of SCR defined a group of patients with significantly higher mortality rate (odds ratio 4.89, 95% confidence interval 2.07 to 11.57). Three simple noninvasive clinical criteria of successful reperfusion may be used to identify a group of patients with poor prognosis after thrombolytic therapy in whom alternative strategies could be applied.     

Inhibition of the delayed rectifier K current in guinea-pig cardiomyocytes by thiamine tetrahydrofurfuryl disulfide

In this study, we attempted to determine which method was the best for reinnervating the laryngeal adductor muscles by comparing nerve suture, nerve implantation, and nerve-muscular pedicle (NMP) transfer, as well as the length of time that could elapse after denervation and still allow for successful reinnervation with the ansa cervicalis. Reinnervation was performed in 36 dogs, at 6-, 8-, 10-, 12- and 18-month intervals after denervation via the three methods of muscle reinnervation described above. We noted some return of adduction in the cases using nerve suture before a 10-month interval after denervation, and with nerve implantation and NMP transfer before the 8-month intervals. The variable adduction was caused by reinnervation of the adductor muscles from the ansa cervicalis, as demonstrated by laryngeal spontaneous and evoked electromyography, the strength of muscle contraction, and histologic findings. Adduction was not observed in the cases after the above-mentioned intervals but partial improvement of the bulk and strength of the reinnervated vocal cord was still achieved. An analysis of the experimental results showed that nerve suture was superior to nerve implantation and the NMP technique. Little difference was noted between nerve implantation and the NMP technique.     

Relationship between internal calcium and outward current in mammalian ventricular muscle; a mechanism for the control of the action potential duration?

In sheep and calf ventricular bundles, increasing the internal calcium by increasing the frequency of voltage-clamping to plateau range potentials increased the time-independent outward current. This effect was more marked with higher [Ca]o, and was reduced if the Ca current blockers Verapamil or D 600 were used. 2. If the internal Ca was increased by the addition of cyanide and reduction of external sodium the outward current was also increased. The frequency-dependent increase in outward current also occurred in this Na-poor (12 mM) solution. 3. Tension measurement on the ventricular bundles showed that a Na-free solution with cyanide did not cause a contracture. On changing from Tyrode to a Na-free solution containing cyanide, and on changing back to Tyrode there was a potentiation of the twitch. 4. In Na-poor solution with cyanide, although no contracture was found, ECa was less positive, suggesting that under these circumstances Ca accumulates at the inner side of the membrane, but not around the myofibrils. 5. The prolongation of the action potential in Cl-free solution is frequency-dependent. A greater prolongation is seen at lower frequencies suggesting that Cl current is relatively more important for repolarization at lower frequencies of stimulation. 6. It is suggested that calcium at the inner side of the membrane sets the level of the background outward current. A feed-back mechanism on this basis is proposed for the control of the action potential duration. Various factors that could influence this basic mechanism are discussed.     

Concentration-dependent block of sodium current in guinea pig ventricular myocytes by a class III antiarrhythmic agent, MS-551

The 32P-post-labelling assay for DNA adduct quantification gives the opportunity to examine endogenous exposure to DNA reactive compounds. Most human biomonitoring studies applied white blood cells (WBC) or cells obtained by broncho-alveolar lavages (BAL) as source of DNA, but still it is not clear what cell type represents the most reliable indicator for exposure to cigarette smoke-associated genotoxins. At first, we examined DNA adduct levels by means of nuclease P1 (NP1) enriched 32P-post-labelling in separated WBC subpopulations after in vitro incubations for 18 h with 10 microM benzo[a]pyrene (B[a]P). DNA adduct levels were highest in monocytes (10.7 +/- 2.9 adducts/10(8) nucleotides, n = 8), followed by lymphocytes (5.9 +/- 1.7, n = 8), and granulocytes (0.5 +/- 0.2, n = 8). Secondly, aromatic-DNA adduct levels were determined in BAL cells and WBC-subsets from (non-)smoking volunteers. In smoking individuals, adduct levels were in the ranking order: BAL cells (3.7 +/- 1.0, n = 5) > monocytes (2.0 +/- 0.5, n = 8) > or = lymphocytes (1.6 +/- 0.4, n = 8) > granulocytes (0.8 +/- 0.2, n = 8) by NP1-enrichment and monocytes (9.0 +/- 3.2, n = 5) > or = lymphocytes (8.0 +/- 2.1, n = 6) > granulocytes (2.1 +/- 0.3, n = 7) by butanol-enriched 32P-post-labelling. Aromatic-DNA adduct levels were significantly higher in WBC-subsets of smokers as compared with non-smokers, except for DNA adducts in granulocytes using butanol enrichment. Thirdly, dose-response relationships were investigated in mononuclear white blood cells (MNC, i.e. monocytes plus lymphocytes) and BAL-cells of a larger group of smoking individuals (n = 78). Adduct levels in MNC were related to daily exposure to cigarette-tar (r = 0.31, P < 0.01). Adduct levels in BAL cells seemed to be correlated with pack-years, but after correction for age this relationship was lost. Butanol extraction resulted in 5-6-fold higher DNA adduct levels in MNC, whereas butanol extraction of BAL-DNA of the same individuals yielded only 2-fold higher adduct levels. The two enrichment procedures of 32P-post-labelling were correlated in BAL cells (r = 0.86, P < 0.001, n = 12). We conclude that particularly MNC are good surrogates for the detection of smoking-related DNA adducts.     

Opposite modulation of 4-aminopyridine and hypoxic hyperexcitability by A1 and A2 adenosine receptor ligands in rat hippocampal slices

A randomly selected, age-stratified sample of subjects 50 years of age and older, living in the Salford Health District area of Greater Manchester was drawn from the age-sex register of a four-doctor group practice and invited by post to enter a study of ptosis. Of 851 subjects approached, 499 (59%) replied. Of these, 99 refused to participate. The remaining 400 were visited at home and underwent a standardized protocol of ophthalmic history, and examination including a photograph of the eyes in the primary position. Forty-six (11.5%) of the subjects had ptosis and its prevalence increased with age. Ptosis was bilateral in 18 (39%) and unilateral in 28 (61%). In all but four cases, the ptosis was acquired. The cause was evident in 23 (50%), with 11 cases being due to mechanical ptosis and 12 to aponeurotic disinsertion secondary to a known pathology. A further 22 cases had primary aponeurotic disinsertion and there was one case of probable myasthenia gravis. The prevalence of pupillary diameter of 1 mm or less increased significantly with age.     

Inhibition by verapamil and diltiazem of agonist-stimulated contractile responses in mammalian ventricular cardiomyocytes

Secretin, vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP) each exert potent positive contractile responses directly in rat ventricular cardiomyocytes. However, the contractile-coupling mechanisms associated with these responses have not been determined. In the present study, the involvement of L-type calcium channels in the contractile responses elicited by each peptide has been investigated using the selective antagonists at L-type calcium channels, verapamil and diltiazem. Ventricular cardiomyocytes, isolated from the hearts of adult rats, were stimulated to contract at 0.5 Hz in the presence of CaCl2 (2 mM) and adenosine deaminase (5U/ml). Cardiomyocytes were pre-incubated for 3 min prior to stimulation, in the absence of L-type calcium channel antagonist, and in the presence of verapamil (< or = 1 microM) or diltiazem (< or = 1 microM). Verapamil (< or = 1 microM) and diltiazem (< or = 1 microM) inhibited the contractile responses elicited by isoprenaline (100 nM) and forskolin (40 microM), used as positive controls, significantly, and in a concentration-dependent manner, but did not inhibit significantly the contractile response elicited by phenylephrine (2 microM), which was employed as a negative control. Verapamil (< or = 1 microM) and diltiazem (< or = 1 microM) inhibited the contractile responses to secretin (20 nM) and VIP (20 nM) significantly, and in a concentration-dependent manner, but did not inhibit the contractile response to CGRP. These data indicate that the positive contractile responses to secretin and VIP in mammalian ventricular cardiomyocytes involve the influx of calcium ion via L-type calcium channels, while the positive contractile response to CGRP does not.