Spatial, cellular and temporal basis of vasopressin potentiation of norepinephrine-induced cAMP formation.

This study investigated the spatial distribution of vasopressin V(1) and beta(1)-adrenoceptors within hippocampal subfields and lamina in an attempt to localize the site(s) of interaction between these two receptor systems. In addition, the cell types, neuronal and glial, in which the vasopressin-induced neuromodulation occurs, were identified. Lastly, the temporal constraints of the potentiation induced by vasopressin were investigated. Results of these analyses demonstrated multiple sites within the hippocampus where the interaction between vasopressin and norephinephrine could occur. Moreover, vasopressin-induced potentiation of adrenergic stimulated cyclase occurred in both hippocampal neurons and glia whereas it did not occur in undifferentiated neurons. Analysis of the temporal constraints of vasopressin-induced potentiation revealed that pre-activation of the vasopressin V(1) receptor for 1 min yielded greater potentiation than simultaneous exposure to vasopressin and norepinephrine. These data provide insights into the spatial and temporal characteristics for the interaction between the vasopressin receptor and adrenoceptor systems and provide a cellular and biochemical rationale for the behavioral findings of Kovács and De Wied.

Fate of the predominant phospholipid component of DepoFoam drug delivery matrix after intrathecal administration of sustained-release encapsulated cytarabine in rats.

The distribution, metabolism, and excretion of dioleoylphosphatidylcholine (DOPC), the predominant phospholipid component of DepoFoam (DF) drug delivery matrix, was determined after lumbar intrathecal injection of double-radiolabeled ((14)CDOPC, (3)H-cytarabine) sustained-release encapsulated cytarabine (DF-cytarabine) in rats prepared with chronic spinal catheters. Radioactivity was quantitated in central nervous system (CNS) and peripheral tissues, cerebrospinal fluid (CSF), blood, urine, and feces at various time points up to 504 h. The distribution of (14)C radiolabel among lipid classes was also determined in selected body fluid samples. Both radiolabels distributed rapidly throughout the neuraxis after injection. Levels of both labels declined in a biphasic manner from CSF and plasma, with an initial rapid decline over the first 96 h, followed by a much slower rate of decline out to 504 h. Greater than 90% of the (3)H (drug) label was estimated to be excreted in urine. In contrast, the data suggest that most of the (14)C (phospholipid) label was expired as (14)CO(2); small percentages of the dose remained incorporated in CNS (7%) and peripheral tissues (8%) or were excreted in urine (6%). Characterization of lipidic (14)C in plasma confirmed metabolism of the parent lipid. The data confirm the sustained-release nature of the DF-cytarabine multivesicular liposomal preparation. Moreover, the results indicate that the DOPC lipid component enters standard catabolic path-ways after breakdown of the DF particles in the intrathecal space. Similar CSF and plasma kinetic profiles of drug and lipid radio-labels support the hypothesis that release of drug is related directly to breakdown of the lipid particles.

Effect of peptidases at the blood brain barrier on the permeability of enkephalin.

The blood brain barrier (BBB) presents an enzymatic barrier to the passage of peptides, from blood to brain. The studies presented here used a well established in vitro model of the BBB to measure the presence of peptidases and the permeability of two opioid peptides. The in vitro BBB model consisted of confluent monolayers of bovine brain microvessel endothelial cells (BMECs). Enkephalin metabolizing enzymes, total aminopeptidase, aminopeptidase M (APM), angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) activities were measured in BMEC monolayers. The effect of specific inhibitors of APM, ACE and NEP on the permeability of [Met5]enkephalin (Met-Enk) and a conformationally constrained and enzymatically stable analog, DPDPE, also was determined. High levels of membrane-associated enzyme activity were measured for total aminopeptidase, APM and ACE. Interestingly, the permeability coefficient of Met-Enk was increased 4-fold in the presence of specific inhibitors of APM and ACE. Low levels of NEP activity were measured in BMEC monolayers and inhibition of NEP had no effect on Met-Enk permeability. The permeability coefficient for DPDPE was not increased with enzyme inhibitors but was 4-fold greater than Met-Enk alone. In the presence of APM or ACE inhibitors, there was no difference in the permeability of DPDPE and Met-Enk. These experiments demonstrate the presence of specific peptidases in BMECs and that the presence of inhibitors to Met-Enk inactivating peptidases significantly increased permeability of this biologically active peptide.

The role of serum testosterone in the accelerated extinction of a conditioned taste aversion in fluid deprived male rats.

Fluid deprivation decreases serum testosterone (T) levels and increases the rate of extinction of a conditioned taste aversion in Sprague-Dawley male rats. It has been suggested that the decreased serum levels may be the primary factor responsible for the accelerated extinction rates during fluid deprivation. To test the generality of this hypothesis, the effect of fluid deprivation on T levels and extinction rate was investigated in Fischer 344 male rats. Extinction rates were accelerated in Fischer 344 rats but T levels were not decreased. In a second study, the behavioral and hormonal responses of Fischer 344 and Sprague-Dawley males to fluid deprivation were compared. Extinction rates were increased in both strains of rat by fluid deprivation, but serum T levels were decreased in fluid-deprived Sprague-Dawley males and not Fischer 344 males. It was suggested that the accelerated extinction in fluid-deprived Sprague-Dawley males was primarily due to decreases in serum T levels, while the faster extinction in deprived Fisher 344 males could be accounted for by decreases in sensitivity to T.

Assessment of an in vitro blood-brain barrier model using several [Met5]enkephalin opioid analogs.

Confluent monolayers of primary and continuous passaged cultures of bovine brain microvessel endothelial cells (BMEC) have been suggested to model the blood-brain barrier (BBB). Increased lipophilicity has been previously suggested to increase BBB penetration. The intent of this study was to examine the effect that structural modifications of the [Met5]enkephalin analog DPDPE had on lipophilicity and passage across the BMEC. The BMEC consisted of a monolayer of confluent primary BMEC grown on polycarbonate (10 microns) filters. Permeability coefficients were calculated on the basis of the diffusion of peptides across the BMEC in a Side-Bi-Side diffusion chamber. Lipophilicity of the peptides examined was determined by using reversed-phase HPLC and calculating the capacity factor (k). Diffusion across the BMEC (for all peptides examined) was linear from 15 to 120 min; therefore, these time points were used to calculate permeability coefficients. Permeability coefficients ranged from 14.34 to 92.00 cm/min (x 10(-4), with [rho-ClPhe4,4']biphalin the highest. Analysis of variance coupled with the Newman-Keuls test showed significantly greater (P < .01) passage of select peptide analogs across the BMEC, including [rho-ClPhe4,4']biphalin, [rho-ClPhe4]DPDPE and reduced DPDPE. Interestingly, upon passage across the confluent monolayer, reduced DPDPE was converted to cyclized DPDPE. Calculated HPLC capacity factors ranged from 3.82 to 12.50. The most lipophilic peptide (highest) examined was acetylated Phe0-DPDPE. Analysis of the regression line of permeability coefficients plotted against capacity factors yielded a correlation coefficient of 0.745 (P < .01). The data provided in this study offer strong evidence that increasing peptide lipophilicity enhances passage across the BMEC.(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased testosterone levels and accelerated extinction of a conditioned taste aversion in fluid-deprived male rats.

The hypothesis that fluid deprivation accelerates extinction of a conditioned taste aversion in male Sprague-Dawley-derived rats by reducing serum testosterone levels was tested. Serum testosterone levels were found to be lower in fluid-deprived males than in nondeprived males (Experiments 1 and 2). Exogenous testosterone treatment that results in high physiological levels of serum testosterone slowed the extinction of fluid-deprived gonadectomized males to rates comparable with those of nondeprived sham males (Experiment 3). It was noted, however, that testosterone treatment was less effective in slowing extinction in fluid-deprived gonadectomized males than in nondeprived gonadectomized males even though the serum testosterone levels were the same (Experiments 3 and 4). These results provide strong support for the original hypothesis, but they suggest that fluid deprivation also reduces sensitivity to testosterone.

Vasopressin-induction of cyclic AMP in cultured hippocampal neurons.

We investigated the influence of AVP on the induction of the second messenger cyclic AMP (cAMP) during early hippocampal neuron development using cultured hippocampal neurons. Results of those studies revealed that in cultured hippocampal neurons AVP-induces the formation of the second messenger cyclic AMP (cAMP). AVP-induction of cAMP is dose dependent and displays an inverted-U shaped function. Maximal AVP-induction of cAMP accumulation occurred following 15 min of exposure. Results of peptide specificity studies indicated that the vasopressin receptor expressed in cultured hippocampal neurons is pharmacologically promiscuous in that vasopressin metabolite peptides, oxytocin, a V2 receptor agonist and antagonist can all induce the formation of cAMP. In marked contrast, [Phe2,Ile3,Orn8]-vasopressin, a V1 receptor agonist, did not induce cAMP formation. The expression of the cAMP-linked AVP receptor is transient with maximal functional expression occurring between 3 and 4 days in culture which recedes by the fifth day in culture. Because the peptide specificity of the cAMP-linked neural AVP receptor is unique, relative to all other AVP receptors studied thus far, we suggest the term V2b receptor to indicate the distinction of a third (3) type of AVP receptor which is expressed during development (D) of hippocampal nerve cells.