Effects of angiotensin II blockade on inflammation-induced alterations of pharmacokinetics and pharmacodynamics of calcium channel blockers.

BACKGROUND AND PURPOSE: Inflammation elevates plasma verapamil concentrations but diminishes pharmacological response. Angiotensin II is a pro-inflammatory mediator. We examined the effect of angiotensin II receptor blockade on the pharmacokinetics and pharmacodynamics of verapamil, as well as the binding properties and amounts of its target protein in calcium channels, in a rat model of inflammation. EXPERIMENTAL APPROACH: We used 4 groups of male Sprague-Dawley rats (220-280 g): inflamed-placebo, inflamed-treated, control-placebo and control-treated. Inflammation as pre-adjuvant arthritis was induced by injecting Mycobacterium butyricum on day 0. From day 6 to 12, 30 mg kg(-1) oral valsartan or placebo was administered twice daily. On day 12, a single oral dose of 25 mg kg(-1) verapamil was administered and prolongation of the PR interval measured and plasma samples collected for verapamil and nor-verapamil analysis. The amounts of the target protein Ca(v)1.2 subunit of L-type calcium channels in heart was measured by Western blotting and ligand binding with (3)H-nitrendipine. KEY RESULTS: Inflammation reduced effects of verapamil, although plasma drug concentrations were increased. This was associated with a reduction in ligand binding capacity and amount of the calcium channel target protein in heart extracts. Valsartan significantly reversed the down-regulating effect of inflammation on verapamil's effects on the PR interval, and the lower level of protein binding and the decreased target protein. CONCLUSIONS AND IMPLICATIONS: Reduced responses to calcium channel blockers in inflammatory conditions appeared to be due to a reduced amount of target protein that was reversed by the angiotensin II antagonist, valsartan.

Nerve growth factor regulates sodium but not potassium channel currents in sympathetic B neurons of adult bullfrogs.

The TTX-sensitive and -resistant components of the voltage-gated Na(+) current (TTX-s I(Na) and TTX-r I(Na)) are increased within 2 wk of cutting the axons of B-cells in bullfrog paravertebral sympathetic ganglia (BFSG). Axotomy also increases the noninactivating, voltage-activated K(+) current (M current I(M)), whereas delayed rectifier K(+) current (I(K)) is reduced. We found that similar effects were produced when BFSG B cells were dissociated from adult bullfrogs and maintained in a defined-medium, neuron-enriched, low-density, serum-free culture. Thus the density of TTX-s I(Na), TTX-r I(Na), and I(M) were transiently increased, whereas I(K) density was decreased. Reduction in voltage-sensitive, Ca(2+)-dependent K(+) current (I(C)) was attributed to previously documented decreases in Ca(2+) channel current (I(Ca)). To test whether axotomy- or culture-induced changes in ion channel function reflect loss of retrograde influence of nerve growth factor (NGF), we examined the effect of murine beta-NGF on TTX-s I(Na), TTX-r I(Na), I(K), and I(M). Culture of neurons for 15 days in the presence of NGF (200 ng/ml), more than doubled total I(Na) density but did not enhance neurite outgrowth. The TTX-r I(Na) density was increased about threefold and the TTX-s I(Na) density increased 2.4-fold. NGF did not affect the activation or inactivation kinetics of the total Na(+) conductance. Effects of NGF were blocked by the transcription inhibitors, cordycepin (20 microM) and actinomycin D (0.01 microg/ml). I(K) and I(M) were unaffected by NGF, and although I(C) was enhanced, this likely reflected the known effect of NGF on I(Ca) in BFSG neurons. Na(+) channel synthesis and/or expression in adult sympathetic neurons is therefore subject to selective regulation by NGF. Despite this, the increase in I(Na) and I(M) as well as the decrease in I(K) seen in BFSG neurons in culture or after axotomy cannot readily be explained in terms of alterations in the availability of target-derived NGF.

Interaction of a semirigid agonist with Torpedo acetylcholine receptor.

The binding of the semirigid agonist [(3)H]arecolone methiodide to the Torpedo nicotinic acetylcholine receptor has been correlated with its functional properties measured both in flux studies with Torpedo membrane vesicles and by single-channel analysis after reconstitution in giant liposomes. Under both equilibrium and preequilibrium conditions, the binding of arecolone methiodide is similar to that of other agonists such as acetylcholine. At equilibrium, it binds to two sites per receptor with high affinity (K(d) = 99 +/- 12 nM), and studies of its dissociation kinetics suggest that each of these sites is made up of two subsites that are mutually exclusive at equilibrium. The kinetics of arecolone methiodide binding were monitored by the changes in the receptor intrinsic fluorescence, and the data are consistent with a model in which the initial binding event is followed by sequential conformational transitions of the receptor-ligand complex. In flux studies, arecolone methiodide was approximately 3-fold more potent (EC(50) = 31 +/- 5 microM) than acetylcholine but its maximum flux rate was 4-10-fold lower. This phenomenon has been studied further by single-channel analysis of Torpedo receptors reconstituted in giant liposomes. Whereas the flexible agonist carbamylcholine (5 microM) was shown to induce channels with conductances of 56 and 34 pS with approximately equal frequency, arecolone methiodide (2 microM) preferentially induced the channel of lower conductance. These results are interpreted in terms of a simple model in which the rigidity of arecolone methiodide restrains the conformation that the receptor-ligand complex can adopt, thus favoring the lower conductance state.

Involvement of Ras/MAP kinase in the regulation of Ca2+ channels in adult bullfrog sympathetic neurons by nerve growth factor.

The cellular mechanisms that underlie nerve growth factor (NGF) induced increase in Ca(2+)-channel current in adult bullfrog sympathetic B-neurons were examined by whole cell recording techniques. Cells were maintained at low density in neuron-enriched, defined-medium, serum-free tissue culture for 6 days in the presence or absence of NGF (200 ng/ml). The increase in Ba2+ current (IBa) density induced by NGF was attenuated by the RNA synthesis inhibitor cordycepin (20 microM), by the DNA transcription inhibitor actinomycin D (0.01 microgram/ml), by inhibitors of Ras isoprenylation (perillic acid 0.1-1.0 mM or alpha-hydroxyfarnesylphosphonic acid 10-100 microM), by tyrosine kinase inhibitors genistein (20 microM) or lavendustin A (1 microM), and by PD98059 (10-100 microM), an inhibitor of mitogen-activated protein kinase kinase. Inhibitors of the phosphatidylinositol 3-kinase (PI3K) pathway (wortmannin, 100 nM, or LY29400, 100 microM) were ineffective as were inhibitors of phospholipase C gamma (U73122 or neomycin, both 100 microM). The effect of NGF persisted in Ca(2+)-free medium that contained 1.8 mM Mg2+ and 2 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. It was mimicked by a Trk antibody that was capable of inducing neurite outgrowth in explant cultures of bullfrog sympathetic ganglion. Antibodies raised against the low-affinity p75 neurotrophin receptor were ineffective in blocking the effect of NGF on IBa. These results suggest that NGF-induced increase in Ca2+ channel current in adult sympathetic neurons results, at least in part, from new channel synthesis after Trk activation of Ras and mitogen activated protein kinase by a mechanism that is independent of extracellular Ca2+.

Regulation of N- and L-type Ca2+ channels in adult frog sympathetic ganglion B cells by nerve growth factor in vitro and in vivo.

To examine mechanisms responsible for the long-term regulation of Ca2+-channels in an adult neuron, changes in whole cell Ba2+ current (IBa) were examined in adult bullfrog sympathetic ganglion B cells in vitro. Cells were cultured at low density in defined, serum free medium. After 15 days, total IBa was similar to the initial value, whereas IBa density was reduced by approximately 36%, presumably due to an increase in neuronal surface area. By contrast, IBa density remained constant after 6-15 days in the presence of murine beta-NGF (200 ng/ml), and total IBa was almost doubled. Inclusion of cytosine arabinoside (Ara-C; 10 microM) to inhibit proliferation of nonneuronal cells, did not affect the survival of neurons in the absence of nerve growth factor (NGF) nor did it attenuate IBa. Ara-C did not prevent the effect of NGF on IBa. There were three independent components to the action of NGF; during 6-9 days, it increased omega-conotoxin-GVIA-sensitive N-type IBa (IBa,N); increased nifedipine-sensitive L-type IBa (IBa,L) and decreased inactivation of the total Ba2+ conductance (gBa). The latter effect involved a selective decrease in the amplitude of one of the four kinetic components that describe the inactivation process. Total IBa was also 55.8% larger than control in the somata of B cells acutely dissociated from leopard frogs that had received prior subcutaneous injections of NGF. By contrast, injection of NGF antiserum decreased total IBa by 29.4%. There was less inactivation of gBa in B cells from NGF-injected animals than in cells from animals injected with NGF antiserum (P < 0.001). These data suggest that NGF-like molecule(s) play(s) a role in the maintenance of IBa in an adult amphibian sympathetic neuron; the presence of NGF may allow the neuron to maintain a constant relationship between cell size and current density. They also show that IBa inactivation in an adult neuron can be modulated in a physiologically relevant way by an extracellular ligand.

Trophic regulation of action potential in bullfrog sympathetic neurones.

These experiments tested the hypothesis that the normal electrophysiological properties of mature bullfrog sympathetic ganglion (BFSG) neurones are maintained by the retrograde supply of nerve growth factor-like molecules from peripheral target tissues. Maintenance of these cells in explant culture in the absence of nerve growth factor (NGF) for up to 30 days produced electrophysiological changes that resemble those previously shown to accompany axotomy in vivo. These included (i) an increase in action potential (ap) duration (spike width), (ii) a decrease in the amplitude of the afterhyperpolarization (ahp), which follows the ap, and (iii) a rapidly developing decrease in ahp duration. When murine NGF (2.5 s; 50 ng/mL) was included in the culture medium there was less attenuation of ahp amplitude. Inclusion of affinity-isolated sheep IgG antibodies (0.5 micrograms/mL; raised against murine 2.5 s NGF) in the culture medium promoted a greater reduction in ahp amplitude than was seen in the "control" explants that were maintained in the absence of NGF. By contrast, the decrease in ahp duration that occurred in control explants was neither attenuated by exposure to NGF nor was it enhanced by NGF antibodies. Also, the increase in spike width that was seen in control explants was enhanced both by murine NGF and by NGF antibodies. Although some of the data support the hypothesis that factor(s) with some similarity to NGF may be synthesized by BFSG in vitro, loss of the retrograde transport of such factors does not explain all aspects of the electrophysiological response to target deprivation and (or) axotomy.

Cardiac mechanical restitution in active and hibernating Richardson's ground squirrel.

The cardiac mechanical restitution was compared in papillary muscles between the active and the hibernating Richardson's ground squirrels at 0.1, 2.8, and 5 mM external Ca2+ concentration [( Ca2+]o). The amplitude of the restitution was significantly higher in hibernating animals between 37 and 7 degrees C at all [Ca2+]o. The first postrest contraction (F1) was highest at 20 degrees C and lower at 37 and 7 degrees C in both groups. The pause duration for maximum F1 was 30 s in active but 10 s in hibernating animals at 37 degrees C and increased to 100 s in both groups at 7 degrees C. The postrest potentiation was eliminated by 10(-6) M ryanodine at 20 degrees C in both groups, and this inhibitory effect was more pronounced in the hibernating group. Together, our results suggest that the activator Ca2+ for excitation-contraction coupling is mainly derived from the sarcoplasmic reticulum (SR) pool in both active and hibernating ground squirrel, and the dependence on SR Ca2+ release via ryanodine-sensitive Ca2+ channels is more marked in the hibernating state. Furthermore, there is no significant difference in sensitivity of the cardiac mechanical restitution to [Ca2+]o between the active and the hibernating condition.

Transduction of the modulatory effect of catecholamines at the mammalian motor neuron terminal.

MEPP frequency (f) was measured in mouse phrenic nerve hemidiaphragm preparations during exposure to adrenoceptor agonist and antagonist drugs. Epinephrine, norepinephrine (NE), and phenylephrine caused a concentration-dependent increase in frequency that was blocked by prazosin but not by yohimbine or nadolol. Isoproterenol had no effect on MEPP(f). The response to NE was not affected by prior incubation of the tissues with pertussis toxin. The response was, however, reduced or abolished by prior exposure to drugs, the actions of which include protein kinase inhibition, and also to a calmodulin inhibitory concentration of W-7. H-7, an inhibitor of protein kinase C and of cyclic nucleotide-dependent kinases, was ineffective. The response to NE was enhanced by 10 mM Li+. The data indicate the existence of a presynaptic alpha 1-adrenoceptor in the motor neuron terminal and suggest that modulation of transmitter release might be mediated by inositol triphosphate liberation, Ca2+ release into the cytosol and activation of a calmodulin-dependent system.

The augmenting action of banana tree juice on skeletal muscle contraction.

An extract obtained from juice expressed from the stem of the plantain banana tree (Musa sapientum L., var. paradisiaca) induces twitch augmentation in skeletal muscles. The mechanism of this action was investigated in the mouse hemi-diaphragm preparation. Directly evoked twitches and potassium induced (K+) contractures were both augmented by the extract. Twitch augmentation was partly dependent on extracellular Ca2+. The action on K(+)-contractures was unaffected by tetrodotoxin, but the rate of relaxation was enhanced in the absence of extracellular calcium (0[Ca2+]o). Muscle contracture induced by high concentrations of extract was also augmented in 0[Ca2+]o and in the presence of the Ca2(+)-channel blocking agent, nifedipine. The time course of the contracture was shortened in 0[Ca2+]o, but not by nifedipine. Nifedipine enhanced the augmenting effect of the extract on twitches but shortened the time-course of this action. In addition, a muscle contracture was superimposed on the twitching muscle at higher concentrations of nifedipine. Manganese, on the other hand, reduced or abolished the augmenting action of the extract. The results are consistent with an action of banana tree juice on the molecule responsible for excitation-contraction coupling in skeletal muscle, resulting in a labilization of intracellular Ca2+.

Postnatal development of adrenergic responsiveness in the rabbit heart.

It is uncertain how changes in the beta-adrenoceptor population influence the contractility of developing heart. To resolve this we have examined postnatal developmental changes in the adrenergic responsiveness of the rabbit heart. The inotropic effect of isoproterenol on isolated left ventricular papillary muscles from rabbits aged 3, 21, and 90 days was compared with the relative number of beta-adrenoceptors at each age measured using [3H]dihydroalprenolol ([3H]DHA) as the specific ligand. The maximum tension developed in response to isoproterenol increases from 37 +/- 7 to 175 +/- 33% above control twitch tension between 3 and 21 days of age; this is followed by a decrease to 68 +/- 12% in the young adult. During this period of development, there is a decline in EC50 towards increased sensitivity. These differences are partially accounted for by an increase in the numbers of specific [3H]DHA binding sites from 17.3 +/- 2.3 to 56.6 +/- 9.9 fmol/mg wet tissue weight from 3 to 21 days, and a subsequent decrease to 32 +/- 4.5 fmol/mg tissue in the young adult. The proportionally larger increase in contractility compared with the number of beta-adrenoceptor binding sites during the first 3 weeks of life is discussed in terms of the developmental changes in the efficacy of coupling between receptor occupancy and contraction.

Isometric contractile properties and caffeine sensitivity of the diaphragm, EDL and soleus in the mouse.

1. Isometric contractile responses and caffeine sensitivity were studied in isolated mouse hemidiaphragm (HD), extensor digitorum longus (EDL) and soleus muscles. 2. The maximum twitch and tetanic tensions developed by the EDL showed a greater temperature dependence than the soleus. The rates of force development and relaxation were more temperature sensitive in the soleus. 3. For the HD, Q10 values for all contractile parameters were intermediate between those of the EDL and the soleus but closer to those of the EDL. 4. The soleus was significantly more sensitive to caffeine than the EDL while the HD showed a response closer to that of the EDL than the soleus. 5. Costal segments of the HD developed force more rapidly but were less caffeine sensitive than the vertebral or sternal segments. 6. The results indicate that the mouse HD exhibits isometric contractile characteristics and caffeine sensitivity more closely resembling the EDL than the soleus but there is a variation in these properties between the different parts of the HD.

Transduction mechanism involving the presynaptic adenosine receptor at mouse motor nerve terminals.

The inhibitory effect of 2-chloroadenosine on spontaneous quantal release of transmitter at the mouse neuromuscular junction was abolished after pretreating tissues either with pertussis toxin (PTX), or with H7, a protein kinase inhibitor. H7 alone caused a fall in miniature endplate potential (MEPP) frequency, but PTX did not. The results are consistent with the hypothesis that rates of neurotransmitter release are directly related to intraterminal cyclic AMP levels, and that these can be reduced by A1 adenosine receptor agonists through the mediation of a Gi protein.

In vitro reinnervation of adult rat muscle fibres by foreign neurons and transformed chromaffin PC12 cells.

Adult rat muscle fibres were dissociated by using collagenase and maintained in culture. One to nine days later, neurons obtained from stages 22-30 Xenopus laevis embryos, or neonatal spinal cord, or pheochromocytoma (PC12) cells treated with nerve growth factor were added. Subsequently, the co-cultures were maintained for up to eight days. Functional synapses were formed with variable efficiency: 12% in rat-Xenopus nerve-muscle co-cultures, 23% in rat-rat and 33% in PC12 co-cultures. Miniature endplate potentials (MEPPs) and currents (MEPCs) were recorded, at frequencies ranging from 0.01 to 0.9 Hz. Their mean amplitude was smaller than in normal mammalian muscles. The rise time and time-constant of decay of MEPCs was about seven to ten times longer than that found in the original muscle, resembling immature synapses. (+)-Tubocurarine abolished the MEPPs in the rat-PC12 neuromuscular junctions. It is concluded that dissociated adult rat muscle fibres retain their ability of being reinnervated, and can form functional synapses with foreign neurons and transformed chromaffin cells.

Sites of action of dihydropyridine drugs in the mouse hemidiaphragm muscle.

The effects of nifedipine and BAY K8644 on directly evoked isometric twitch and potassium (K+)- and caffeine-induced contractures were investigated in mouse hemidiaphragm preparations in which neuromuscular transmission had been irreversibly blocked. Both drugs caused initial potentiation of twitch which at high concentrations (greater than 3 x 10(-5) M) was followed by blockade. A simultaneous slow contracture was seen with nifedipine but not BAY K8644. Control K+ contractures were triphasic. The initial fast and slow phases of this contracture were potentiated by BAY K8644 at all times and concentrations. Both phases were potentiated by nifedipine at low concentrations but, during prolonged exposure to high concentrations, potentiation was replaced by an inhibition. The time course of activation and inactivation of the slow phase was also accelerated by all concentrations of nifedipine. The initial phase of caffeine-induced contracture was potentiated and resolved into two components. From these results at least three sites of action were postulated. Conventional binding to t-tubular Ca2+ channels was linked to effects on the slow phase of K+ contracture. An effect on Ca2+ release from the sarcoplasmic reticulum and an inhibition of Ca2+ transfer from uptake to release compartments in the sarcoplasmic reticulum are also postulated.

Pharmacological elevation of cyclic AMP and transmitter release at the mouse neuromuscular junction.

Intracellular recordings of spontaneous and evoked end-plate potentials have been made at the neuromuscular junction of mouse hemidiaphragms to determine a possible role of cyclic AMP (cAMP) in the release of acetylcholine from presynaptic terminals. Spontaneous release, as determined from the frequency of miniature end-plate potentials, was increased by drugs that inhibit phosphodiesterase: isobutylmethylxanthine (IBMX), SQ 20,009, theophylline, and caffeine; drugs that stimulate adenylate cyclase: forskolin, fluoride, and cholera toxin, and the stable analogue of cAMP: 8-bromo-cAMP but not dibutyryl cAMP. Release increased with time during maintained exposure to the drugs and generally followed a simple exponential time course with time constants ranging from 8 to 17 min at 20 degrees C, except for SQ 20,009 and cholera toxin which required longer exposure times for effect. The order of potency of the phosphodiesterase inhibitors was IBMX = SQ 20,009 greater than theophylline = caffeine. This is consistent with an effect mediated by an increase in cAMP concentrations within the nerve terminal. Evoked release, determined from the quantal content of the end-plate potential, was increased to a lesser extent than spontaneous release. The results are discussed with reference to the possible involvement of second messengers in the release of vesicles from nerve terminals in vertebrate synapses.

Potassium contractures in the mouse diaphragm: regional variation in the multiphasic response.

1. Submaximal potassium-induced contractures were studied in isolated mouse hemidiaphragms (HD) and compared with extensor digitorum longus (EDL) and soleus. 2. In the HD, the contracture was triphasic: an initial small, fast phase followed by a larger slow phase, and a late phase which was seen on prolonged exposure. 3. Costal segments of the HD responded to elevated potassium with a larger fast phase, while dorsal or ventral segments produced a larger slow phase. 4. In calcium-free solution, the fast phase appeared to be unaffected, while the slow phase was potentiated and its time course abbreviated. The late phase was greatly attenuated or abolished. 5. In the soleus, but not the EDL muscle, contracture amplitude in calcium-free solution was potentiated and the time course was shortened. 6. The characteristics of potassium-induced contractures of both the soleus and EDL muscles are to be seen in the response of the hemidiaphragm, but there is a regional variation in their proportional contribution which suggests a heterogeneity in diaphragm composition.

The inhibitory effects of some adenosine analogues on transmitter release at the mammalian neuromuscular junction.

An electrophysiological study was made of the effects of four adenosine analogues, 2-chloroadenosine (2-CIA), 5'-N-ethylcarboxamidoadenosine (NECA), L-N6-phenylisopropyladenosine (L-PIA), and 2-(p-methoxyphenyl)-adenosine (CV-1674) on neurotransmitter release in the mouse phrenic nerve - hemidiaphragm preparation. All four drugs decreased miniature end-plate potential frequency in a dose-dependent manner. Evoked transmitter release in the cut diaphragm preparation was depressed by 2-CIA and CV-1674 to a similar extent. The ability of theophylline to antagonize the inhibitory effect of CV-1674 on spontaneous transmitter release was also established. On the basis of these results, the rank order of potencies was: L-PIA greater than NECA greater than 2-CIA greater than CV-1674. A clear classification of receptor type could not be made, since the ratio of potencies of L-PIA and NECA was narrow. Different slopes of the concentration-effect curves for 2-CIA and CV-1674 compared with L-PIA and NECA suggest an additional component to simple agonist action in their overall effects.

Diet and cardiac arrhythmia: effects of lipids on age-related changes in myocardial function in the rat.

Male rats were fed for 3-4 months (short-term) or 12-15 months (long-term) on a standard laboratory diet alone (control) or supplemented with sunflower seed oil (SSO, 12% w/w) or sheep kidney fat (SKF, 12% w/w). Papillary muscles were electrically driven (1 Hz, 5 ms, supramaximal voltage) at 37 degrees C in Krebs-Henseleit solution, and contractions were measured isometrically. Both the positive inotropic responses to CA++ and the incidence of spontaneous tachyarrhythmias under catecholamine stress were increased by short-term SKF feeding and with age in control and SKF groups, whereas SSO prevented these changes. The results show a marked effect of age upon ventricular myocardial function in the rat, which appears to be accelerated by the consumption of animal (saturated) fat while polyunsaturated vegetable oil provides some degree of protection. It is suggested that changes in membrane lipid composition can alter the Ca++ handling characteristics of myocardial cells.