Pharmacokinetics of meloxicam after intravenous, intramuscular and oral administration of a single dose to African grey parrots (Psittacus erithacus).

Meloxicam is a nonsteroidal anti-inflammatory drug commonly used in avian species. In this study, the pharmacokinetic parameters for meloxicam were determined following single intravenous (i.v.), intramuscular (i.m.) and oral (p.o.) administrations of the drug (1 mg/kg·b.w.) in adult African grey parrots (Psittacus erithacus; n = 6). Serial plasma samples were collected and meloxicam concentrations were determined using a validated high-performance liquid chromatography assay. A noncompartmental pharmacokinetic analysis was performed. No undesirable side effects were observed during the study. After i.v. administration, the volume of distribution, clearance and elimination half-life were 90.6 ± 4.1 mL/kg, 2.18 ± 0.25 mL/h/kg and 31.4 ± 4.6 h, respectively. The peak mean ± SD plasma concentration was 8.32 ± 0.95 µg/mL at 30 min after i.m. administration. Oral administration resulted in a slower absorption (tmax  = 13.2 ± 3.5 h; Cmax  = 4.69 ± 0.75 µg/mL) and a lower bioavailability (38.1 ± 3.6%) than for i.m. (78.4 ± 5.5%) route. At 24 h, concentrations were 5.90 ± 0.28 µg/mL for i.v., 4.59 ± 0.36 µg/mL for i.m. and 3.21 ± 0.34 µg/mL for p.o. administrations and were higher than those published for Hispaniolan Amazon parrots at 12 h with predicted analgesic effects.

Long-lasting concentrations of cefovecin after subcutaneous and intramuscular administration to Patagonian sea lions (Otaria flavescens).

Cefovecin is a third-generation cephalosporin developed as an aqueous solution for use by the subcutaneous route in dogs and cats. This study evaluated the duration of cefovecin plasma concentrations after single intramuscular (IM) or subcutaneous (SC) injection at different doses in 10 Patagonian sea lions (Otaria flavescens). Blood samples were collected serially from the day of the injection up to 60-90 days post-injection. Plasma drug concentrations were determined by high performance liquid chromatography-UV detection and pharmacokinetic parameters were calculated by non-compartmental analysis. No reactions or side effects associated with the drug were observed in any of the studied animals. Both routes showed very similar pharmacokinetic behaviour. Elimination half-life (11.3-21.6 days, SC; 13.1-15.9 days, IM) and mean residence time (17.6-36.8 days SC; 16.5-25.4 days IM) were, in all cases and doses, considerably longer than those previously reported for any other species. Based on these findings, and preliminary data on specific pathogen sensitivity, cefovecin was found to be a very promising antimicrobial for Patagonian sea lions, in particular those that are difficult to access or that are under certain rehabilitation conditions.

Otilonium: a potent blocker of neuronal nicotinic ACh receptors in bovine chromaffin cells.

1. Otilonium, a clinically useful spasmolytic, behaves as a potent blocker of neuronal nicotinic acetylcholine receptors (AChR) as well as a mild wide-spectrum Ca2+ channel blocker in bovine adrenal chromaffin cells. 2. 45Ca2+ uptake into chromaffin cells stimulated with high K+ (70 mM, 1 min) was blocked by otilonium with an IC50 of 7.6 microM. The drug inhibited the 45Ca2+ uptake stimulated by the nicotinic AChR agonist, dimethylphenylpiperazinium (DMPP) with a 79 fold higher potency (IC50 = 0.096 microM). 3. Whole-cell Ba2+ currents (IBa) through Ca2+ channels of voltage-clamped chromaffin cells were blocked by otilonium with an IC50 of 6.4 microM, very close to that of K(+)-evoked 45Ca2+ uptake. Blockade developed in 10-20 s, almost as a single step and was rapidly and almost fully reversible. 4. Whole-cell nicotinic AChR-mediated currents (250 ms pulses of 100 microM DMPP) applied at 30 s intervals were blocked by otilonium in a concentration-dependent manner, showing an IC50 of 0.36 microM. Blockade was induced in a step-wise manner. Wash out of otilonium allowed a slow recovery of the current, also in discrete steps. 5. In experiments with recordings in the same cells of whole-cell IDMPP, Na+ currents (INa) and Ca2+ currents (ICa), 1 microM otilonium blocked 87% IDMPP, 7% INa and 13% ICa. 6. Otilonium inhibited the K(+)-evoked catecholamine secretory response of superfused bovine chromaffin cells with an IC50 of 10 microM, very close to the IC50 for blockade of K(+)-induced 45Ca2+ uptake and IBa. 7. Otilonium inhibited the secretory responses induced by 10 s pulses of 50 microM DMPP with an IC50 of 7.4 nM. Hexamethonium blocked the DMPP-evoked responses with an IC50 of 29.8 microM, 4,000 fold higher than that of otilonium. 8. In conclusion, otilonium is a potent blocker of nicotinic AChR-mediated responses. The drugs also blocked various subtypes of neuronal voltage-dependent Ca2+ channels at a considerably lower potency. Na+ channels were unaffected by otilonium. This extraordinary potency of otilonium in blocking nicotinic AChR, unrecognised until now, might account in part for its well known spasmolytic effects.

Blocking effects of otilonium on Ca2+ channels and secretion in rat chromaffin cells.

We describe here the effects of otilonium bromide (an anticholinergic agent widely used as an intestinal spasmolytic) on whole-cell currents through Ca2+ channels (IBa) and catecholamine secretion in rat adrenal glands and isolated rat chromaffin cells. Otilonium blocked the peak IBa current in voltage-clamped chromaffin cells in a concentration-dependent manner; the IC50 to block IBa was 4.7 microM. Blockade was not accompanied by a significant shift in the I-V relationship for IBa, suggesting that such blockade was not affecting a specific subtype of Ca2+ channel. When given intracellularly through the patch pipette, otilonium (10 microM) did not block IBa. However, its external application to the same cell (10 microM) reversibly reduced IBa by 70%. Otilonium caused a concentration-dependent blockade of catecholamine release from perfused rat adrenal glands intermittently stimulated with methacholine, high K+ or histamine. The IC50 to block secretion after a 5 min incubation with otilonium was 0.02, 0.7 and 3 microM, respectively, for methacholine, K+ and histamine. The blocking effects of otilonium were fully reversible at concentrations below 10 microM. The Ca2+ channel agonist Bay K 8644 (methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyr idine-5- carboxylate) partially antagonized the effects of otilonium on K(+)-evoked secretion and accelerated the time course of recovery from inhibition. The results are compatible with the idea that otilonium blocks Ca2+ entry into chromaffin cells by blocking voltage-dependent Ca2+ channels. This would lead to a limitation in the rise in cytosolic Ca2+ at secretory sites and to inhibition of catecholamine release in response to stimulation of chromaffin cells.

Respiring mitochondria determine the pattern of activation and inactivation of the store-operated Ca(2+) current I(CRAC).

In eukaryotic cells, hormones and neurotransmitters that engage the phosphoinositide pathway evoke a biphasic increase in intracellular free Ca(2+) concentration: an initial transient release of Ca(2+) from intracellular stores is followed by a sustained phase of Ca(2+) influx. This influx is generally store dependent. Most attention has focused on the link between the endoplasmic reticulum and store-operated Ca(2+) channels in the plasma membrane. Here, we describe that respiring mitochondria are also essential for the activation of macroscopic store-operated Ca(2+) currents under physiological conditions of weak intracellular Ca(2+) buffering. We further show that Ca(2+)-dependent slow inactivation of Ca(2+) influx, a widespread but poorly understood phenomenon, is regulated by mitochondrial buffering of cytosolic Ca(2+). Thus, by enabling macroscopic store-operated Ca(2+) current to activate, and then by controlling its extent and duration, mitochondria play a crucial role in all stages of store-operated Ca(2+) influx. Store-operated Ca(2+) entry reflects a dynamic interplay between endoplasmic reticulum, mitochondria and plasma membrane.

Energized mitochondria increase the dynamic range over which inositol 1,4,5-trisphosphate activates store-operated calcium influx.

In eukaryotic cells, activation of cell surface receptors that couple to the phosphoinositide pathway evokes a biphasic increase in intracellular free Ca2+ concentration: an initial transient phase reflecting Ca2+ release from intracellular stores, followed by a plateau phase due to Ca2+ influx. A major component of this Ca2+ influx is store-dependent and often can be measured directly as the Ca2+ release-activated Ca2+ current (I(CRAC)). Under physiological conditions of weak intracellular Ca2+ buffering, respiring mitochondria play a central role in store-operated Ca2+ influx. They determine whether macroscopic I(CRAC) activates or not, to what extent and for how long. Here we describe an additional role for energized mitochondria: they reduce the amount of inositol 1,4,5-trisphosphate (InsP3) that is required to activate I(CRAC). By increasing the sensitivity of store-operated influx to InsP3, respiring mitochondria will determine whether modest levels of stimulation are capable of evoking Ca2+ entry or not. Mitochondrial Ca2+ buffering therefore increases the dynamic range of concentrations over which the InsP3 is able to function as the physiological messenger that triggers the activation of store-operated Ca2+ influx.

Extracellular ATP regulates exocytosis in inhibiting multiple Ca(2+) channel types in bovine chromaffin cells.

Feedback modulation of voltage-dependent Ca2+ channels by ATP is a well documented phenomenon in bovine chromaffin cells. However, its influence in the control of hormone release is at present poorly understood. By using combined patch-clamp and fura-2 fluorescence measurements we provide evidence that the three Ca2+ channel types (L, N and P/Q) expressed in bovine chromaffin cells are inhibited by ATP (30 microM), and that their involvement in the secretory response, as assayed by capacitance measurements, is roughly proportional to their contribution to the whole-cell Ca2+ current (ICa) both in the absence and presence of ATP. ATP did not modify the capacitance increase observed in cells dialyzed with Ca(2+)-EGTA buffers (1.5 microM free Ca2+), thus excluding a direct effect of ATP on the secretory machinery. Voltage predepolarizations or long chemical (2 s, 70 mM KCl) depolarizations attenuate the effect of ATP on exocytosis by partially relieving the inhibition of ICa Likewise, a strong stimulation that depletes the readily releasable pool of vesicles prevents an inhibitory effect of ATP on the secretory response. While these results lend support to the hypothesis of autocrine modulation of exocytosis by endogenously released ATP acting on P2y-purinoceptors to inhibit ICa, feedback regulation of the rate of release will be a complex function of the occupancy of those receptors and of the electrical and secretory activity of the cell.

Characterization of adrenal medullary chromaffin cells by flow cytometry.

BACKGROUND: Adrenomedullary chromaffin cells are neural crest derivatives widely used as a model system to study neurosecretory mechanisms. Morphological, immunohistochemical, and functional data indicate that chromaffin cells are heterogeneous and support the distinction between adrenaline (A)- and noradrenaline (NA)-producing and secreting cells. The aim of this study was to characterize by flow cytometry the two main chromaffin cell subtypes in suspensions of cultured bovine chromaffin cells. METHODS: An indirect immunofluorescence method was used for the specific labeling of two intracellular enzymes, dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT), involved in the synthesis of NA and A, respectively. Flow cytometry analysis of fluorescence labeling was performed in two chromaffin cell fractions differentially enriched in A-containing cells by centrifugation through density gradients. PNMT and DBH-related fluorescence was also correlated with the A and NA content of the cells assayed by HPLC measurements. RESULTS: No significant differences were found in forward-side scatter plots between the two cell fractions (A-enriched cells and mixed cells); however, the degree of labeling of the enzymes and the corresponding PNMT/DBH-related fluorescence ratio was significantly greater in the A-enriched cell fraction. The existence of changes in DBH and PNMT content of chromaffin cells over time (1 week) in culture was also examined. No significant variation in enzyme related fluorescence values was detected in any of the two cell fractions, and this result correlated well with HPLC determinations of the catecholamine content (A and NA) of the cells. CONCLUSIONS: Flow cytometry appears to be a useful technique to characterize chromaffin cell subtypes and to follow their phenotypic changes in response to growth factors.

The mechanism of calcium channel facilitation in bovine chromaffin cells.

1. This study was planned to clarify the mechanism of Ca2+ channel facilitation by depolarizing prepulses given to voltage-clamped bovine chromaffin cells. The hypothesis for an autocrine modulation of such channels was tested by studying the effects of a soluble vesicle lysate (SVL) on whole-cell Ba2+ currents (IBa). 2. SVL was prepared from a bovine adrenal medullary homogenate. The ATP content in this concentrated SVL amounted to 3.18 +/- 0.12 mM (n = 4). The concentration of noradrenaline and adrenaline present in the SVL was 11.2 +/- 0.97 and 15.2 +/- 2 mM, respectively (n = 5). A 1:1000 dilution of SVL in the external solution halved the magnitude of IBa and produced a 7-fold slowing of its activation kinetics. The blocking effects of SVL were concentration dependent and quickly reversed upon washout. 3. Inhibition and slowing of the kinetics of IBa by SVL could be partially reversed by strong depolarizing prepulses (+90 mV, 45 ms). This reversal of inhibition, called Ca2+ channel facilitation, persisted in the presence of 3 microM nifedipine. 4. Intracellular dialysis of GDP-beta-S (0.5 mM) or pretreatment of the cells with pertussis toxin (100 ng ml-1 for 18-24 h) prevented the reduction in peak current caused by a 1:100 dilution of SVL; no prepulse facilitation could be observed under these conditions. 5. The receptor blockers naloxone (10 microM) or suramin (100 microM) and PPADS (100 microM) largely antagonized the effects of SVL. Treatment of SVL with alkaline phosphatase or dialysis against a saline buffer to remove low molecular mass materials (< 10 kDa) considerably reduced the activity of SVL. 6. Stopping the flow of the external solution (10 mM Ba2+) gradually reduced the size, and slowed down the activation phase, of the current. Prepulse facilitation of IBa was absent or weak in a superfused cell, but was massive upon flow-stop conditions in the presence or absence of 3 microM nifedipine. 7. Our experiments suggest that facilitation by prepulses of whole-cell current through Ca2+ channels is due to the suppression of an autoinhibitory autocrine loop present in bovine chromaffin cells. By acting at least on purinergic and opiate receptors, the exocytotic release of ATP and opiates will cause a tonic inhibition of the current through a G-protein-mediated mechanism. Such a mechanism will be removed by strong depolarizing prepulses, and will involve preferentially non-L-type channels. In the light of these and other recent results, previously held views on the selective recruitment by prepulses of dihydropyridine-sensitive Ca2+ channels are not tenable.