Norman Weiner - US grants

The acute activation of tyrosine hydroxylase from central catecholaminergic neurons, peripheral adrenergic neurons and adrenal medulla following nerve stimulation, stress and administration of drugs known to affect nervous function will be characterized further. Employing monospecific antibody to tyrosine hydroxylase and gel electrophoresis procedures which permit the rapid isolation of tyrosine hydroxylase from tissues, attempts will be made to determine the precise stoichiometry of the phosphorylation of the enzyme and the relation between phosphorylation and enzyme activation. The role of calcium-dependent regulator protein (calmodulin, CDR) and calcium in activation of tyrosine hydroxylase will be assessed. The effects of nerve stimulation, cyclic AMP-dependent protein kinase, phospholipid and anion activation of the enzyme will be compared in order to determine whether the molecular basis for the activation is analogous in all circumstances. Protein kinases and phosphoprotein phosphatases from adrenergic tissues will be purified and characterized and their possible relevance to the activation and deactivation of tyrosine hydroxylase will be determined. The role of cyclic AMP-independent protein kinase in the regulation of tyrosine hydroxylase in central and peripheral adrenergic tissues will be evaluated. The mechanism of the induction of tyrosine hydroxylase in vivo during chronic stress and in PC12 and neuroblastoma cells in culture after exposure to cyclic nucleotides, glucocorticoids or chronic stress depolarizing conditions will be examined further. Ultrastructural localization of tyrosine hydroxylase will be determined by immunocytochemical techniques and the anatomical relationship of tyrosine hydroxylase containing nerve terminals to cholinergic, gabaergic and peptidergic nerve terminals in the brain will be examined. Regulation of catecholamine release consequent to nerve stimulation from brain slices, brain synaptosomes and the isolated vas deferens preparation will be evaluated with particular emphasis on cyclic nucleotides, opioid agonists, alpha adrenoceptor, beta adrenoceptor, dopamine and cholinergic agonists. The relationship between tyrosine hydroxylase activation and dopamine release in amacrine cells of the retina of the rabbit following photic stimulation or electrical stimulation in situ will be determined. The uptake of tyrosine into adrenergic (Text Truncated - Exceeds Capacity)

Infections caused by the herpes virus are among the oldest known to man. Recurrent herpes simplex labialis has been reported to affect almost one-half of the population of the United States, and 25 per cent of those affected have frequent and/or severe recurrences. Genital herpes, though not officially recognized as a disease until 1966, is currently an epidemic venereal disease. About five million Americans suffer from this problem, with an estimated one-half million individuals having become infected in 1981 alone. Although many forms of therapy have been tested, none has proven profoundly beneficial in decreasing the severity and frequency of the clinical manifestations. Systemic regimens adequate to suppress skin symptomology often result in adverse systemic effects and still may not overcome the inaccessibility to the drug of the target tissue. "Drug delivery" remains the singularly most limiting factor to the effective treatment of herpes. Preliminary studies showing that interferon can be delivered across intact skin when incorporated in liposomes suggest a means of treating certain skin infections. The goal of these studies is to assess the possibility of treating virus-infected epithelial cells with liposomally entrapped interferon delivered by the transdermal route. Specifically, we wish to isolate and elucidate the factors associated with the liposomal entrapment of biologically active interferon and with its subsequent delivery through intact skin to tissue infected by virus. Since interferon's biological activity is long-lasting when in liposomes, liposomes also appear to offer a practical as well as an effective means of delivery of interferon to lesion sites. Furthermore, a systematic analysis of the parameters involved may provide a rational hypothesis and guidance for future research on the delivery of liposomal interferon by other routes of administration for the treatment of other disease states.

Determination of the precise mechanisms of ethanol's actions on CNS neurotransmitter function has been the focus of much ongoing investigative effort. Use of an animal model of genetic differences in initial CNS sensitivity to ethanol (LS and SS mice) has provided evidence that certain of the behavioral actions of ethanol may be mediated by this agent's differential effects on monoaminergic neuronal systems in specific brain regions and the adrenal gland. It has also been determined that LS and SS mice differ substantially in the extent of the analeptic response to thyrotropin-releasing hormone (TRH) following ethanol administration. Although controversy exists as to the exact sites and mechanism of TRH's analeptic action it is believed to involve activation of central cholinergic and/or monoaminergic neurons. A thorough examination of TRH effects on monoamine function in LS and SS mice in the presence and absence of ethanol should provide further insight into the neurochemical mechanisms mediating both the CNS depression by ethanol and its antagonism by TRH. Similar experiments will be conducted with pentobarbital, a CNS depressant to which LS and SS mice do not differ in initial CNS sensitivity, and the results compared to those obtained with ethanol. Initial studies will include a comparison of the effects of TRH with various TRH analogs (possessing improved pharmacokinetic and non-endocrine profiles) on behavioral actions of ethanol or pentobarbital, i.e., narcosis and hypothermia. Subsequently, the effects of TRH and TRH analogs on these behavioral measures of ethanol and pentobarbital intoxication will be correlated with their effects on ethanol-induced alterations of monoamine neurochemistry. Aspects of monoamine function to be evaluated in brain regions and adrenal gland include: (1) monoamine synthesis by performing in vitro kinetic analysis of the enzymes tyrosine and tryptophan hydroxylase as well as in vivo estimates of enzyme activity by measuring the accumulation of dopa and 5-hydroxytryptophan, respectively, following treatment with a decarboxylase inhibitor, (2) levels of the enzyme substrates, tyrosine and tryptophan, (3) availability of the enzyme cofactor by measuring biopterin levels and the activity of its synthetic enzyme, GTP-cyclohydrolase, and (4) monoamine release (overflow) in vivo in specific brain regions by employing both electrochemical and microdialysis techniques. Finally, in order to assess the possible contribution of the pituitary-thyroid axis to the analeptic and thermogenic actions of acute TRH and TRH analog administration, we will evaluate their effects on thyroid function, including serum thyrotropin (TSH) triiodothyronine (T3), and thyroxine (T4) levels, % hormone uptake, and free hormone index, in the presence and absence of ethanol and pentobarbital.

Genital herpes, though not officially recognized as a disease until 1966, is currently an epidemic veneral disease. About five million Americans suffer from this problem, with an estimated one-half million individuals becoming infected each year. Although many forms of forms of therapy have been tested, none has proven profoundly beneficial in decreasing the severity and frequency of the clinical manifestations. Once established, herpes virus particles retreat into the nerve trunk and remain latent in the asymptomatic period. They are inaccessible to therapy in their ganglionic home. Therefore, control of herpes labialis rests on the ability we have to suppress symptoms promptly as the virus begins to re- express itself. It appears this could best be done by delivering an effective antiviral agent into the living epidermal tissues of the skin during the prodromal stage of lesion development. Interferon seems to have especially high potential for the treatment of herpes, condylomata acuminata and other similarly manifested disease states. However, systemic regimens adequate to suppress skin symptomology often results in adverse systemic effects and still may not overcome the inaccessibility of the target tissue to the drug. "Drug delivery" remains the singularly most limiting factor to the effective treatment of herpes. Liposomes recently have received much attention in the search for a more effective means of delivering intrinsically active drugs to their tissue targets. Recent studies in our laboratories and others have shown that large amounts of interferon are taken up by liposomes, that significant amounts of this interferon are internalized within the liposome, that liposomally entrapped interferon is stable for at least one year, and that interferon maintains its antiviral activity under these conditions. In recent studies, we have shown that liposomes facilitate skin deposition of drugs and that when interferon is encapsulated in a liposome by a technique which facilitates its association with bilayers, the polypeptide penetrates intact skin and is therapeutically active; however, interferon incorporated in traditional vehicles lack therapeutic efficacy. The goal of these studies is to continue to assess the possibility of treating virus-infected epithelial cells with liposomally entrapped interferon delivered by the transdermal route. Specifically, we wish to isolate and elucidate the factors related to the liposomal entrapment of biologically active interferon and with its subsequent delivery through intact skin to tissue infected by virus. Since interferon's biological activity is long-lasting when in liposomes, liposomes also appear to offer a practical as well as an effective means of delivery of interferon to lesion sits. Furthermore, a systematic analysis of the parameters involved may provide a rational hypothesis and guidance for future research on the topical delivery of other liposomally entrapped drugs for the treatment of other disease states.