Calcium channel blockers and behavioral sensitization

Behavioral sensitization to amphetamine-induced stereotypy was previously shown to consist of two separable phenomena, induction and expression, both of which involve the excitatory amino acids (EAA). In the present experiments, the calcium channel blockers (CCB), nifedipine, diltiazem and verapamil, were shown to block both phenomena; these results are similar to those reported earlier for DNQX, an antagonist of the non-N-methyl-D-aspartate receptors for the EAA. The CCB, like DNQX, affect only that percentage of the stereotypic response which results from the sensitization reaction, without affecting the quantitative portion of the response attributable to the acute effect of amphetamine. The results support previous conclusions that the sensitization response consists of two quantitative components, only one of which involves the EAA. The antagonism exhibited by the CCB suggests that behavioral sensitization involves Ca++ and L-type calcium channels.     

Calcium channel blockers inhibit bacterial chemotaxis

The effect of several Ca2+ channel blockers, which inhibit the voltage-dependent Ca2+ uptake in Bacillus subtilis, on chemotactic behaviour of the bacterium was studied. Nitrendipine, verapamil, LaCl3 and omega-conotoxin were tested and these blockers inhibited chemotactic behaviour in the bacterium toward L-alanine. Among these blockers, omega-conotoxin was the most effective inhibitor of chemotaxis. EGTA was also as effective as omega-conotoxin. In contrast, these blockers, did not inhibit the motility and the growth of the bacterium. These results suggest that internal Ca2+ plays an important role in the sensory system of bacterial chemotaxis.     

Impaired generation of prostaglandins from isolated gastric surface epithelial cells in portal hypertensive rats

We studied generation of prostaglandins E2 and 6-keto F1a by surface epithelial cell isolated from the gastric mucosa of portal hypertensive and sham-operated rats. Oxygenated cell suspensions containing 80 +/- 3% of surface epithelial cells were incubated for 30 min at 37 degrees C and the concentration of prostaglandin E2 and 6-keto-prostaglandin F1a in medium was measured by radioimmunoassay. Viability of the cells was assessed with Fast green exclusion at baseline and after 30-min and 60-min incubation. Within 30 minutes the surface epithelial cells obtained from portal hypertensive rats generated 22.0 +/- 1.6 (mean +/- SE) pg prostaglandin E2 and 40.7 +/- 4.7 pg 6-keto prostaglandin F1a, per 10(6) cells. These were significantly less than prostaglandin generation by cells obtained from sham-operated rats. The viability of the surface epithelial cells from portal hypertensive rats was also significantly reduced compared with sham-operated rats after 60 minute incubation. Reduced ability of the surface epithelial cells to generate prostaglandins may be one mechanism for increased susceptibility of portal hypertensive gastric mucosa to injury by noxious agents.     

Ca2+ channel blockers inhibit secretory C1-channels in intestinal epithelial cells.

Outwardly rectifying Cl- channels are present in the human colonic cell line (HT29D4). The classical Cl- channel blocker 5-nitro-2(3-phenylpropylamino)benzoate inhibits Cl- channel activity with a K0.5 value of 20 microM. Epithelial Cl- channel activity is inhibited by Ca2+ channel blockers. Phenylalkylamines are the most effective inhibitors. (+/-)Verapamil and (-)desmethoxyverapamil induce flickering and then the complete blockade of Cl- channels recorded from outside-out patches. K0.5 values are 60 microM and 100 microM for (-)desmethoxyverapamil and (+/-)verapamil, respectively. Other classes of L-type Ca2+ channel blockers have also been studied but they are less active.     

Calcium channel blockers inhibit endogenous pyrogen fever in rats and rabbits.

We have previously shown that febrile responses in both rats and rabbits are elicited by the intravenous injection of a semipurified endogenous pyrogen (EP) prepared from human monocytes. We are now presenting evidence that these febrile responses are mediated via activation of Ca2+ channels by EP. The febrile responses of male New Zealand White rabbits and Sprague-Dawley rats to a standard dose of EP were determined at their respective thermoneutral ambient temperatures. The animals were then treated with Ca2+ channel blocker verapamil (7.5 mg/kg iv) 30-60 min before the EP challenge. In every case the febrile response to EP was markedly attenuated after verapamil pretreatment, while administration of verapamil by itself had no detectable effect on body temperature. Another Ca2+ channel blocker, nifedipine (5 mg/kg iv), was shown to possess antipyretic activity in rats also. To localize where in the fever pathway these Ca2+ channel blockers were acting, we investigated the effect of verapamil at the same dose on fevers that were produced by microinjection of prostaglandin E (PGE) directly into the brain. These PGE fevers were unaffected by verapamil pretreatment, indicating that the antipyretic action of Ca2+ channel blockers occurs before the formation of PGE in response to EP stimulation. The most likely locus of action is the activation of the enzyme phospholipase A2, which regulates the production of arachidonic acid from cellular phospholipids in the prostanoid cascade.     

Calcium channel blockers inhibit galvanotaxis in human keratinocytes

Directed migration of keratinocytes is essential for wound healing. The migration of human keratinocytes in vitro is strongly influenced by the presence of a physiological electric field and these cells migrate towards the negative pole of such a field (galvanotaxis). We have previously shown that the depletion of extracellular calcium blocks the directional migration of cultured human keratinocytes in an electric field (Fang et al., 1998; J Invest Dermatol 111:751-756). Here we further investigate the role of calcium influx on the directionality and migration speed of keratinocytes during electric field exposure with the use of Ca(2+) channel blockers. A constant, physiological electric field strength of 100 mV/mm was imposed on the cultured cells for 1 h. To determine the role of calcium influx during galvanotaxis we tested the effects of the voltage-dependent cation channel blockers, verapamil and amiloride, as well as the inorganic Ca(2+) channel blockers, Ni(2+) and Gd(3+) and the Ca(2+) substitute, Sr(2+), on the speed and directionality of keratinocyte migration during galvanotaxis. Neither amiloride (10 microM) nor verapamil (10 microM) had any effect on the galvanotaxis response. Therefore, calcium influx through amiloride-sensitive channels is not required for galvanotaxis, and membrane depolarization via K(+) channel activity is also not required. In contrast, Sr(2+) (5 mM), Ni(2+) (1-5 mM), and Gd(3+) (100 microM) all significantly inhibit the directional migratory response to some degree. While Sr(2+) strongly inhibits directed migration, the cells exhibit nearly normal migration speeds. These findings suggest that calcium influx through Ca(2+) channels is required for directed migration of keratinocytes during galvanotaxis and that directional migration and migration speed are probably controlled by separate mechanisms.     

Calcium channel blockers in acute myocardial infarction and unstable angina: an overview.

OBJECTIVE--To assess the effects of calcium channel blockers on development of infarcts, reinfarction, and mortality. DESIGN--A systematic overview of all randomised trials of calcium channel blockers in myocardial infarction and unstable angina. PATIENTS--19,000 Patients in 28 randomised trials. RESULTS--In the trials of myocardial infarction 873 deaths occurred among 8870 patients randomised to active treatment compared with 825 deaths among 8889 control patients (odds ratio of 1.06, 95% confidence interval of 0.96 to 1.18). There was no evidence of a beneficial effect on development and size of infarcts or rate of reinfarction. The results were similar in short term trials in which treatment was confined to the acute phase and those in which treatment was started some weeks later and continued for a year or two. There was no evidence of heterogeneity among different calcium channel blockers in their effects on any end point. The results were similar in the unstable angina trials (110 out of 561 patients treated with calcium channel blocker compared with 104 out of 548 controls developed a myocardial infarction; 14 out of 591 treated compared with nine out of 578 controls died). CONCLUSIONS--Calcium channel blockers do not reduce the risk of initial or recurrent infarction or death when given routinely to patients with acute myocardial infarction or unstable angina.     

Polarized expression of CD74 by gastric epithelial cells.

CD74 is known as the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) that regulates the cell biology and functions of MHC class II molecules. Class II MHC and Ii expression was believed to be restricted to classical antigen-presenting cells (APC); however, during inflammation, other cell types, including mucosal epithelial cells, have also been reported to express class II MHC molecules. Given the importance of Ii in the biology of class II MHC, we sought to examine the expression of Ii by gastric epithelial cells (GEC) to determine whether class II MHC molecules in these nonconventional APC cells were under the control of Ii and to further support the role that these cells may play in local immune and inflammatory responses during Helicobacter pylori infection. Thus we examined the expression of Ii on GEC from human biopsy samples and then confirmed this observation using independent methods on several GEC lines. The mRNA for Ii was detected by RT-PCR, and the various protein isoforms were also detected. Interestingly, these cells have a high level expression of surface Ii, which is polarized to the apical surface. These studies are the first to demonstrate the constitutive expression of Ii by human GEC.     

Secretory phospholipase A2 mediates cooperative prostaglandin generation by growth factor and cytokine independently of preceding cytosolic phospholipase A2 expression in rat gastric epithelial cells

Transforming growth factor (TGF)-alpha and interleukin (IL)-1beta are responsible for the healing of gastric lesions through, in part, prostaglandin (PG) generation. We examined the contribution of cytosolic and secretory phospholipase A(2)s (cPLA(2) and sPLA(2)) to the PG generation by rat gastric epithelial cells in response to both stimuli. Stimulation with TGF-alpha for 24 h increased cPLA(2) and cyclooxygenase (COX)-2 markedly, PGE(2) slightly, and type IIA sPLA(2) and COX-1 not at all, whereas IL-1beta increased sPLA(2) only. Both stimuli synergistically increased PGE(2), sPLA(2), and the two COXs but not cPLA(2). The onset of the PGE(2) generation paralleled the sPLA(2) release but was apparently preceded by increases in cPLA(2) and the two COXs. The increase in PGE(2) was impaired by inhibitors for sPLA(2) and COX-2 but not COX-1. cPLA(2) inhibitors suppressed PGE(2) generation by TGF-alpha alone but not augmentation of PGE(2) generation or sPLA(2) release by IL-1beta in combination with TGF-alpha. Furthermore, despite an increase in cPLA(2) including its phosphorylated form (phosphoserine), -induced arachidonic acid liberation was impaired in the TGF-alpha/IL-1beta-stimulated cells, in which p11, a putative cPLA(2) inhibitory molecule, was also increased and co-immunoprecipitated with cPLA(2). These results suggest that synergistic stimulation of sPLA(2) and COX-2 expression by TGF-alpha and IL-1beta results in an increase in PGE(2). Presumably, the preceding cPLA(2) expression is not involved in the PGE(2) generation, because of impairment of its hydrolytic activity in the stimulated cells.     

Calcium channel blockers stimulate LDL receptor synthesis in human skin fibroblasts

Human skin fibroblasts incubated in lipoprotein-deficient medium in the presence of 50-100 microM of the calcium channel blockers verapamil or diltiazem incorporated up to 2.5 times more [35S]methionine into immunoprecipitable LDL receptor protein than did control cells. Verapamil was found to be more potent in this regard than diltiazem. The calcium channel blockers did not influence the overall synthesis of cellular proteins or the half-life of the LDL receptor, and they were not able to prevent the suppression of LDL receptor synthesis caused by exogenous LDL or 25-hydroxycholesterol. The calcium channel blocker-induced stimulation of LDL receptor synthesis was accompanied by a corresponding increase in binding and internalization of [125I]LDL, but the degradation of internalized lipoprotein was slightly decreased. The results suggest that intracellular Ca2+ levels modulate LDL receptor metabolism in human skin fibroblasts.     

Calcium channel blockers modulate airway constriction in the canine lung periphery

We studied the effect of two voltage-sensitive calcium channel blockers on Na2EDTA-induced bronchoconstriction in the canine lung periphery. A wedged bronchoscope technique was used to measure collateral system resistance before and after challenges with aerosolized Na2EDTA, hypocapnia, aerosolized acetylcholine, and increased flow of dry air in anesthetized mongrel dogs. Nifedipine, a dihydropyridine calcium channel blocker, reduced hypocapnia-induced bronchoconstriction by 88 +/- 6% (SE) but did not alter Na2EDTA-induced constriction. Verapamil, a phenylalkylamine calcium channel blocker, attenuated hypocapnia- and Na2EDTA-induced bronchoconstriction by 69 +/- 6 and 44 +/- 7%, respectively, but did not significantly alter responses to either acetylcholine or dry air challenge. We conclude that calcium influx through voltage-sensitive calcium channels, perhaps of the T subtype, has a limited role in the initiation of Na2EDTA-induced bronchoconstriction in the canine lung periphery.     

Calcium homeostasis of epithelial cells

1. Cytosolic free Ca2+ is an important regulator of ion transport processes in epithelial cells. 2. Free Ca2+ concentration is regulated by a concerted action of Ca2+ transport systems in plasma membranes and intracellular organelles. 3. These transport systems were studied in intestinal and renal cortical cells with emphasis on the transport capacities and Ca2+ affinities. 4. Ca2+ accumulation by permeabilized cells was compared to Ca2+ uptake by isolated organelles and membrane fractions. 5. Effects induced by cell or organelle isolation methods and the influence of temperature and pH on Ca2+ transport capacities were studied.     

Interaction of human neutrophils with airway epithelial cells: Reduction of leukotriene B4 generation by epithelial cell derived prostaglandin E2

Airway epithelial cells (AEC) play an active role in the regulation of inflammatory airway disease. In the present study we analyzed the interaction of AEC with polymorphonuclear leukocytes (PMN) in coincubation with respect to their arachidonic acid (AA) metabolism using reversed phase-HPLC and post-HPLC-ELISA. Primary cultures of porcine AEC released predominantly PGE₂, PGF_2a, and 15-hydroxyeicosatetraenoic acid (15-HETE), whereas the major human PMN-derived AA metabolite was the chemotactic factor leukotriene B₄ (LTB₄). In AEC-PMN cocultures stimulated with the calcium ionophore A23187, PMN-related 5-lipoxygenase products were decreased by 45%. This reduction in LTB₄ formation in the presence of AEC was mainly due to PGE₂ generated by the epithelial cells, whereas 15-HETE made a minor contribution. Most of the effect was inhibited by AEC pretreatment with acetylsalicylic acid and restored by addition of equivalent amounts of exogenous PGE₂. LTB₄ degradation was not enhanced in PMN-AEC coincubations. Moreover, reduction of LTB₄ formation in this system did not require an intimate cell-to-cell contact as shown by studies involving filter membranes for PMN-AEC separation. Superoxide anion concentrations were also decreased in PMN-AEC coincubations; this effect, however, was unrelated to PGE₂ for quantitative reasons and was probably due to 2 is the major mediator in the coincubation of porcine AEC and human PMN that downregulates neutrophil responses by activating receptors on the neutrophil. A minor contributor in this course of PMN-AEC interaction may be the 15-HETE transcellular pathway. Overall, airway epithelium appears to play an antiinflammatory role by damping the proinflammatory potential of neutrophils. J. Cell. Physiol. 175:268–275, 1998. © 1998 Wiley-Liss, Inc.