Slow frequency-dependence of action potential afterhyperpolarization in bullfrog sympathetic ganglion neurones

The afterhyperpolarization (AHP) which follows the action potential (AP) in bullfrog sympathetic ganglion B-cells involves activation of Ca²⁺-sensitive K⁺ conductances following Ca²⁺ influx via Ca²⁺ channels. The duration of AHPs evoked at 2-s stimulus intervals were 70.05±3.76% of those evoked at 90-s stimulus intervals (n=35). Since there was no consistent effect of ryanodine (5 M), ruthenium red, (300 M) or dantrolene Na (35 M) on this frequency dependence, it is unlikely to result from release of Ca²⁺ from intracellular stores. Ca²⁺ currents (I _Ca, studied by means of the whole-cell patch-clamp technique, exhibited a slow frequency dependence as a result of a slow inactivation process which was independent of Ca²⁺-induced I _Ca inactivation and I _Ca run-down. There was excellent correlation (r=0.964) between the estimated changes in Ca²⁺ influx and the expected activation of the Ca²⁺-sensitive K⁺ current, I _AHP. This result is consistent with the hypothesis that the frequency dependence of the AHP is a consequence of the slow inactivation of I _Ca.     

Influence of caffeine,Ca2+, and Mg2+ on ryanodine depression of the tension transient in skinned myocardial fibers of the rabbit

Ryanodine, a blocker for Ca²⁺-release channels of the sarcoplasmic reticulum (SR Ca²⁺-release channels), induces depression of myocardial contraction in isolated intact muscle, which is consistent with depression of the caffeine-induced tension transient in skinned muscle fibers. In isolated SR, ryanodine binds to a specific receptor with high affinity, and this binding is enhanced by caffeine and increasing Ca²⁺ and decreased by increasing Mg²⁺. The aim of this study was to test the hypothesis that depression of myocardial contraction is mediated by changes in ryanodine-receptor binding properties. Accordingly, factors (caffeine, Ca²⁺, and Mg²⁺) affecting ryanodine-receptor binding properties in the isolated SR membrane were studied in skinned myocardial fibers from adult rabbits. The depression of the caffeine-induced tension transient by ryanodine (ryanodine depression) influenced by these three factors was measured. In a dose-dependent manner, increasing caffeine or Ca²⁺ concentrations enhanced the ryanodine depression. The concentrations for 50% ryanodine depression (IC₅₀) approximated 7mM for caffeine, and pCa 5.25 for Ca²⁺. When 1 M ryanodine and 25 mM caffeine were combined, ryanodine depression was independent of Ca²⁺ at low Ca²⁺ concentrations (20%–30% at pCa>8 and 7.5) and was a direct function of Ca²⁺ at higher concentrations (pCa 7.5–6.0 with IC₅₀ approx. pCa 6.75). In contrast, increasing Mg²⁺ reduced the ryanodine depression with IC₅₀ approximately equal to pMg 3.3. In conclusion, the caffeineor Ca²⁺-enhanced, and Mg²⁺-reduced ryanodine depression observed in this study is consistent with known ryanodinereceptor binding properties.     

Molecular and functional characterization of a mutant allele of the mitogen-activated protein-kinase geneSLT2(MPK1) rescued from yeast autolytic mutants

 We have further characterized the functionality of the Saccharomyces cerevisiae gene SLT2(MPK1), coding for a MAP-kinase homolog essential for cell integrity, which is involved in the Pkc1p signalling pathway. This gene was isolated on the basis of its capacity to complement the thermosensitive-autolytic, osmotic-remediable phenotype of lyt2 mutants. Both slt2Δ and lyt2 mutants displayed a caffeine-sensitive phenotype consisting of cell lysis that was not dependent on temperature. Caffeine concentrations affecting the growth of these mutant strains were dependent on the genetic background, the SSD1 allele being very significant in this regard. The SLT2 allele of several lyt2 strains was both rescued and amplified by PCR. The recovered allele was shown to be non-functional as it could not complement the lytic phenotype of both deletion (slt2Δ) and lyt2 strains. After nucleotide sequencing of the recovered allele, we found that the defect of lyt2 mutants consists in a substitution of an aspartic acid for a glycine at position 35 of the amino-acid sequence of Slt2p. Gly35 is the third glycine of a glycine cluster (Gly-X-Gly-X-X-Gly), a conserved region in protein kinases and other nucleotide-binding proteins. Received: 13 October/27 November 1995     

Caffeine-induced oscillations of cytosolic Ca2+ in GH3 pituitary cells are not due to Ca2+ release from intracellular stores but to enhanced Ca2+ influx through voltage-gated Ca2+ channels

Caffeine, a well known facilitator of Ca²⁺-induced Ca²⁺ release, induced oscillations of cytosolic free Ca²⁺ ([Ca²⁺]_i) in GH₃ pituitary cells. These oscillations were dependent on the presence of extracellular Ca²⁺ and blocked by dihydropyridines, suggesting that they are due to Ca²⁺ entry through L-type Ca²⁺ channels, rather than to Ca²⁺ release from the intracellular Ca²⁺ stores. Emptying the stores by treatment with ionomycin or thapsigargin did not prevent the caffeine-induced [Ca²⁺]_i oscillations. Treatment with caffeine occluded phase 2 ([Ca²⁺]_i oscillations) of the action of thyrotropin-releasing hormone (TRH) without modifying phase 1 (Ca²⁺ release from the intracellular stores). Caffeine also inhibited the [Ca²⁺]_i increase induced by depolarization with high-K⁺ solutions (56% at 20 mM), suggesting direct inhibition of the Ca²⁺ entry through voltage-gated Ca²⁺ channels. We propose that the [Ca²⁺]_i increase induced by caffeine in GH₃ cells takes place by a mechanism similar to that of TRH, i.e. membrane depolarization that increases the firing frequency of action potentials. The increase of the electrical activity overcomes the direct inhibitory effect on voltage-gated Ca²⁺ channels with the result of increased Ca²⁺ entry and a rise in [Ca²⁺]_i. Consideration of this action cautions interpretation of previous experiments in which caffeine was assumed to increase [Ca²⁺]_i only by facilitating the release of Ca²⁺ from intracellular Ca²⁺ stores.     

Caffeine induces periodic oscillations of Ca2+-activated K+ current in pulmonary arterial smooth muscle cells

The periodic oscillations of outward currents were studied in smooth muscle cells of the rabbit pulmonary artery. The combined stimuli of superfusion with 1 mM caffeine and depolarization of the membrane potential to 0 mV evoked periodic oscillations of outward currents with fairly uniform amplitudes and intervals. The oscillating outward currents induced by caffeine were dependent on intracellular Ca²⁺ concentration ([Ca²⁺]_i) and had a reversal potential near to the equilibrium potential for K⁺. So the oscillating outward currents are carried by K⁺ through Ca²⁺-dependent K⁺ channels (I _K(Ca)), and may reflect the oscillations of [Ca²⁺]_i. The oscillating outward currents were abolished, or their frequency reduced, by lowering external [Ca²⁺], Ca²⁺ channel blockers, or by 1 M ryanodine, indicating that: (1) there is a continuous influx of Ca²⁺ through the plasma membrane at a holding potential of 0 mV; (2) the periodic transient increases of [Ca²⁺]_i are ascribed to the rhythmic release of Ca²⁺ from ryanodinesensitive intracellular store by the mechanism of Ca²⁺-induced Ca²⁺ release (CICR). On the basis of the above results, we simulated the oscillation of [Ca²⁺]_i induced by caffeine, which is known to lower the threshold of CICR. The patterns of peak amplitude histograms of spontaneous transient outward currents (STOC) in the oscillating cells were different from those in non-oscillating cells. The amplitudes of STOC in the latter were more variable than those in the former. The oscillating outward currents were modulated by 1 M forskolin and 1 M sodium nitroprusside, but STOC were little affected. The above differences between STOC and oscillating outward currents suggest that the two currents are activated by the Ca²⁺ originating from different intracellular Ca²⁺ stores which are functionally heterogeneous.     

A case of “myopathy with tubular aggregates” with increased muscle fibre sensitivity to caffeine

Summary A 23-year-old man with myopathy with tubular aggregates had suffered from exercise-induced muscle cramps for 1 year. His general and neurological findings were normal. Laboratory investigations were within normal limits except for a slightly elevated serum creatine kinase level. Muscle biopsy showed some small angular fibres and scattered type 2B fibres with prominent tubular aggregates originating from the sarcoplasmic reticulum. Since the muscle fibres contracted at a lower concentration of caffeine, increased muscle fibre sensitivity to caffeine is probably related to muscle cramps in this disorder. Tubular aggregates are then secondarily formed in the muscle fibres.     

Pharmacologic specificity of tolerance to caffeine-induced stimulation of locomotor activity

The pharmacologic specificity of tolerance to caffeine-induced stimulation of locomotor activity was studied in adult male rats that were given access to either caffeine solution (0.5 or 1.0 mg/ml) or plain water for 10 min every 6 h on a chronic daily basis; daily caffeine intake averaged 41 and 62 mg/kg, respectively. Dose-effect curves were determined for behavioral stimulant and depressant drugs in control and caffeine-treated groups. Drugs were injected IP and locomotor activity was measured for 30 min beginning 35 min later. Rats tolerant to stimulation of locomotor activity by caffeine were also tolerant to theophylline and 7-(2-chloroethyl)theophylline, but not to any of six nonxanthine stimulants, including cocaine, methylphenidate, and d-amphetamine. The adenosine analogs, R(–)-N⁶-2-(phenylisopropyl)adenosine(R(–)-PIA) and 5-(N-ethyl)carboxamidoadenosine (NECA), decreased locomotor activity of control and caffeine-treated (0.5 mg/ml) rats; dose-effect curves in rats consuming caffeine chronically were displaced to the right of the control curves by 10-fold for R(–)-PIA and 100-fold for NECA. Dose-effect curves for the nonadenosine behavioral depressants chlorpromazine and diazepam were unchanged by chronic treatment with caffeine, but the curve for pentobarbital, which is thought to inhibit adenosine receptor binding, was shifted to the right by a factor of 3. Rats withdrawn from chronic caffeine for 24 h were still completely tolerant to caffeine-induced stimulation of locomotor activity. Dose-effect curves for R(–)-PIA and d-amphetamine in rats withdrawn from chronic caffeine for 24 h were not different from curves in control animals. These results indicate that tolerance to caffeine-induced stimulation of locomotor activity is specific to the methylxanthine class of stimulants and is not a property of nonxanthine psychomotor stimulants. Furthermore, the adenosine-antagonist activity of caffeine remains evident even in rats completely tolerant to the stimulant effect of caffeine. These results provide no support for the view that caffeine tolerance is due to enhanced sensitivity of central adenosine systems.A preliminary report of this work appeared in The Pharmacologist (28:140, 1986)     

Analytical studies on illicit heroin

The recovery of heroin in fumes was investigated. In the Netherlands the common mode of heroin smoking is the chasing the dragon procedure: heroin is heated on an aluminium foil by a lighter and the fumes are inhaled. The efficiency of the volatilization of heroin using this procedure was studied under laboratory conditions using thin layer chromatography, gas chromatography and high pressure liquid chromatography. A considerable influence of the form (salt or base) of the heroin was found as well as strong influences of other substances that may be present in illicit heroin samples as diluents. The danger of the inhalation of fumes containing unknown pyrolysis products is mentioned and a hypothesis is given for the phenomenon of heroin-leucoencephalopathy that was observed in heroin smokers in Amsterdam in 1981. The types of heroin encountered in the Netherlands are discussed with regard to their suitability for smoking.     

Caffeine actions on currents induced by calcium-overload in Purkinje fibers

The ionic events underlying the changes induced by caffeine in calcium-overloaded Purkinje fibers were studied by means of a voltage-clamp technique. The following results were obtained. In fibers exposed to strophanthidin (5 X 10(-7) M), a depolarizing clamp of suitable magnitude or duration is followed by an oscillatory current (Ios) often superimposed on a decaying inward tail current (the "tail current"). Caffeine (9 mM) abolishes Ios and increases the tail current. Caffeine has similar actions when calcium overload is induced by increasing [Ca]0 or decreasing [Na]0. The magnitude of the tail current is reduced by decreasing [Ca]0. The tail current appears with repolarizations to -40 mV or more negative values as Ios appears in the absence of caffeine. As with Ios the tail current can be triggered twice (during and after a test clamp of suitable characteristics), becomes more inward with repeated clamps and becomes larger with larger or longer conditioning clamps. During the recovery from caffeine exposure, the tail current decreases gradually as Ios returns progressively. It is concluded that both Ios and tail current are caused by calcium overload but are affected in opposite direction by caffeine, apparently because caffeine decreases the calcium overload in the sarcoplasmic reticulum (abolition of Ios) and increases the calcium to be extruded from the sarcoplasm (increase in the tail current).     

The action of caffeine on inward barium current through voltage-dependent calcium channels in single rabbit ear artery cells

The effect of caffeine on inward current carried by barium ions through voltage-dependent calcium channels has been investigated in single rabbit ear artery cells using whole-cell voltage-clamp techniques. Caffeine (1 –30 mM) caused a rapid and reversible concentration-dependent blockade of barium current and a related compound, 3-isobutyl-1-methylxanthine (IBMX), was a more potent inhibitor of barium current. Caffeine-induced inhibition of barium current showed no voltage- or usedependence and caffeine did not alter the steady-state inactivation of barium current. The effect of caffeine was not blocked by extracellular or by intracellular ryanodine or inclusion of both 5 mM 1,2-bis(2-aminophenoxy)-ethane N,N,N,N,-tetraacetic acid (BAPTA) and 2 mM ethylene glycol-bis(-amino ethyl ether) N,N,N,N,-tetraacetic acid (EGTA) in the intracellular solution. Rolipram and M&B 22984, non-xanthine inhibitors of phosphodiesterase, did not diminish inward barium current. The data indicate that caffeine and IBMX block voltage-operated calcium channels and it is suggested that this is due to a direct interaction of methylxanthines with the calcium channel.