Reversal of myocyte hypertrophy by ventricular unloading: cardiac improvement without adrenergic receptor up-regulation and relocalization.

In previous studies, we found that the improved contractile ability of cardiac myocytes from patients who have had left ventricular assist device (LVAD) support was due to a number of beneficial changes, most notably in calcium handling (increased sarcoplasmic reticulum calcium binding and uptake), improved integrity of cell membranes due to phospholipid reconstruction (reduced lysophospholipid content), and an upregulation of adrenoreceptors (increased adrenoreceptor numbers). However, in the case presented here, there was no increase in adrenoreceptor number, which is something that we usually find in core tissue at the time of LVAD removal or organ transplantation; also, there was no homogeneous postassist device receptor distribution. However, the patient was well maintained for 10 months following LVAD implantation, until a donor organ was available, regardless of the lack of adrenoreceptor improvement. We conclude from these studies that cardiac recovery is the result of the initiation of multiple repair mechanisms, and that the lack of expected changes, in this case increased adrenoreceptors, is not always an accurate indicator of anticipated outcome. We suggest that interventions and strategies have to consider multiple, beneficial changes due to unloading and target a number of biochemical and structural areas to produce improvement, even if not all of these improvements occur.

Effects of variable Gs expression on $\beta\sb2$-adrenergic receptor stimulated adenylyl cyclase response

An exact knowledge of the kinetic nature of the interaction between the stimulatory G protein (G$\sb{\rm s}$) and the adenylyl cyclase catalytic unit (C) is essential for interpreting the effects of Gs mutations and expression levels on cellular response to a wide variety of hormones, drugs, and neurotransmitters. In particular, insight as to the association of these proteins could lead to progress in tumor biology where single spontaneous mutations in G proteins have been associated with the formation of tumors (118). The question this work attempts to answer is whether the adenylyl cyclase activation by epinephrine stimulated $\beta\sb2$-adrenergic receptors occurs via G$\sb{\rm s}$ proteins by a G$\sb{\rm s}$ to C shuttle or G$\sb{\rm s}$-C precoupled mechanism. The two forms of activation are distinguishable by the effect of G$\sb{\rm s}$ levels on epinephrine stimulated EC50 values for cyclase activation.^ We have made stable transfectants of S49 cyc$\sp-$ cells with the gene for the $\alpha$ protein of G$\sb{\rm s}$ $(\alpha\sb{\rm s})$ which is under the control of the mouse mammary tumor virus LTR promoter (110). Expression of G$\sb{\rm s}\alpha$ was then controlled by incubation of the cells for various times with 5 $\mu$M dexamethasone. Expression of G$\sb{\rm s}\alpha$ led to the appearance of GTP shifts in the competitive binding of epinephrine with $\sp{125}$ICYP to the $\beta$-adrenergic receptors and to agonist dependent adenylyl cyclase activity. High expression of G$\sb{\rm s}\alpha$ resulted in lower EC50's for the adenylyl cyclase activity in response to epinephrine than did low expression. By kinetic modelling, this result is consistent with the existence of a shuttle mechanism for adenylyl cyclase activation by hormones.^ One item of concern that remains to be addressed is the extent to which activation of adenylyl cyclase occurs by a "pure" shuttle mechanism. Kinetic and biochemical experiments by other investigators have revealed that adenylyl cyclase activation, by hormones, may occur via a Gs-C precoupled mechanism (80, 94, 97). Activation of adenylyl cyclase, therefore, probably does not occur by either a pure "'Shuttle" or "Gs-C Precoupled" mechanism, but rather by a "Hybrid" mechanism. The extent to which either the shuttle or precoupled mechanism contributes to hormone stimulated adenylyl cyclase activity is the subject of on-going research. ^

Molecular mechanisms of protein kinase-mediated desensitization and internalization of the $\beta$-adrenergic receptor

The $\beta$-adrenergic receptor ($\beta$AR), which couples to G$\sb{\rm s}$ and activates adenylylcyclase, has been a prototype for studying the activation and desensitization of G-protein-coupled receptors. The main objective of the present study is to elucidate the molecular mechanisms of protein kinase-mediated desensitization and internalization of the $\beta$AR.^ Activation of cAPK or PKC causes a rapid desensitization of $\beta$AR stimulation of adenylylcyclase in L cells, which previous studies suggest involves the cAPK/PKC consensus phosphorylation site in the third intracellular loop of the $\beta$AR, RRSSK$\sp{263}$. To determine the role of the individual serines in the cAPK- and PKC-meditated desensitizations, wild type (WT) and mutant $\beta$ARs containing the substitutions, Ser$\sp{261} \to$ A, Ser$\sp{262} \to$ A, Ser$\sp{262} \to$ D, and Ser$\sp{261/262} \to$ A, were constructed and stably transfected into L cells. The cAPK-mediated desensitization was decreased 70-80% by the Ser$\sp{262} \to$ A, Ser$\sp{262} \to$ D, and the Ser$\sp{261/262} \to$ A mutations, but was not altered by the Ser$\sp{261} \to$ A substitution, demonstrating that Ser$\sp{262}$ was the primary site of the cAPK-induced desensitization. The PMA/PKC-induced desensitization was unaffected by either of the single serine to alanine substitutions, but was reduced 80% by the double serine to alanine substitution, suggesting that either serine was sufficient to confer the PKC-mediated desensitization. Coincident stimulation of cAPK and PKC caused an additive desensitization which was significantly reduced (80%) only by the double substitution mutation. Quantitative evaluation of the coupling efficiencies and the GTP-shift of the WT and mutant receptors demonstrated that only one of the mutants, Ser$\sp{262} \to$ A, was partially uncoupled. The Ser$\sp{262} \to$ D mutation did not significantly uncouple, demonstrating that introducing a negative charge did not appear to mimic the desensitized state of the receptor.^ To accomplish the in vivo phosphorylation of the $\beta$AR, we used two epitope-modified $\beta$ARs, hemagglutinin-tagged $\beta$AR (HA-$\beta$AR) and 6 histidine-tagged $\beta$AR (6His-$\beta$AR), for a high efficiency purification of the $\beta$AR. Neither HA-$\beta$AR nor 6His-$\beta$AR altered activation and desensitization of the $\beta$AR significantly as compared to unmodified wild type $\beta$AR. 61% recovery of ICYP-labeled $\beta$AR was obtained with Ni-NTA column chromatography.^ The truncation 354 mutant $\beta$AR(T354), lacking putative $\beta$ARK site(s), displayed a normal epinephrine stimulation of adenylylcyclase. Although 1.0 $\mu$M epinephrine induced 60% less desensitization in T354 as compared to wild type $\beta$AR, 1.0 $\mu$M epinephrine-mediated desensitization in T354 was 35% greater than PGE$\sb1$-mediated desensitization, which is essentially identical in both WT and T354. These results suggested that sequences downstream of residue 354 may play a role in homologous desensitization and that internalization may be attributed to the additional desensitization besides the cAMP mechanism in T354 $\beta$AR. (Abstract shortened by UMI.) ^

The differential expression of the $\beta\sb2$-adrenergic receptor modifies agonist-dependent adenylylcyclase activation

There have been multiple reports which indicate that variations in $\beta$AR expression affect the V$\sb{\rm max}$ observed for the agonist-dependent activation of adenylylcyclase. This observation has been ignored by most researchers when V$\sb{\rm max}$ values obtained for wild type and mutant receptors are compared. Such an imprecise analysis may lead to erroneous conclusions concerning the ability of a receptor to activate adenylylcyclase. Equations were derived from the Cassel-Selinger model of GTPase activity and Tolkovsky and Levitzki's Collision Coupling model which predict that the EC$\sb{50}$ and V$\sb{\rm max}$ for the activation of adenylylcyclase are a function of receptor number. Experimental results for L cell clones in which either hamster or human $\beta$AR were transfected at varying levels showed that EC$\sb{50}$ decreases and V$\sb{\rm max}$ increases as receptor number increases. Comparison of these results with simulations obtained from the equations describing EC$\sb{50}$ and V$\sb{\rm max}$ showed a close correlation. This documents that the kinetic parameters of adenylylcyclase activation change with the level of receptor expression and relates this phenomenon to a theoretical framework concerning the mechanisms involved in $\beta$AR signal transduction.^ One of the terms used in the equations which expressed the EC$\sb{50}$ and V$\sb{\rm max}$ as a function of receptor number is coupling efficiency, defined as $\rm k\sb1/k\sb{-1}$. Calculation of $\rm k\sb1/k\sb{-1}$ can be accomplished for wild type receptors with the easily measured experimental values of agonist K$\sb{\rm d}$, EC$\sb{50}$ and receptor number. This was demonstrated for hamster $\beta$AR which yielded a coupling efficiency of 0.15 $\pm$ 0.003 and human $\beta$AR which yielded a coupling efficiency of 0.90 $\pm$ 0.031. $\rm k\sb1/k\sb{-1}$ replaces the traditional qualitative evaluation of the ability to activate adenylylcyclase, which utilizes V$\sb{\rm max}$ without correction for variation in receptor number, with a quantitative definition that more accurately describes the ability of $\beta$AR to couple to G$\sb{\rm s}$.^ The equations which express the EC$\sb{50}$ and V$\sb{\rm max}$ for adenylylcyclase activation as a function of receptor number and coupling efficiency were tested to determine whether they could accurately simulate the changes seen in these parameters during desensitization. Data from original desensitization experiments and data from the literature (24,25,52,54,83) were compared to simulated changes in EC$\sb{50}$ and V$\sb{\rm max}$. In a variety of systems the predictions of the equations were consistent with the changes observed in EC$\sb{50}$ and V$\sb{\rm max}$. In addition reductions in the calculated value of $\rm k\sb1/k\sb{-1}$ was shown to correlate well with $\beta$AR phosphorylation and to be minimally affected by sequestration and down-regulation. ^

$\beta\sb2$-adrenergic receptor desensitization, internalization and phosphorylation in response to full and partial agonists

The objective of this study is to test the hypothesis that partial agonists produce less desensitization because they generate less of the active conformation of the $\beta\sb2$-adrenergic receptor ($\beta$AR) (R*) and in turn cause less $\beta$AR phosphorylation by beta adrenergic receptor kinase ($\beta$ARK) and less $\beta$AR internalization. In the present work, rates of desensitization, internalization, and phosphorylation caused by a series of $\beta$AR agonists were correlated with a quantitative measure, defined as coupling efficiency, of agonist-dependent $\beta$AR activation of adenylyl cyclase. These studies were preformed in HEK-293 cells overexpressing the $\beta$AR with hemagglutinin (HA) and 6-histidine (6HIS) epitopes introduced into the N- and C-termini respectively. Agonists chosen provided a 95-fold range of coupling efficiencies, and, relative to epinephrine, the best agonist, (100%) were fenoterol (42%), albuterol (4.9%), dobutamine (2.5%) and ephedrine (1.1%). At concentrations of these agonists yielding $>$90% receptor occupancy, the rate and extent of the rapid phase (0-30 min) of agonist induced desensitization of adenylyl cyclase followed the same order as coupling efficiency, that is, epinephrine $\ge$ fitnoterol $>$ albuterol $>$ dobutamine $>$ ephedrine. The rate of internalization, measured by a loss of surface receptors during desensitization, with respect to these agonists also followed the same order as the desensitization and exhibited a slight lag. Like desensitization and internalization, $\beta$AR phosphorylation exhibited a dependency on agonist strength. The two strongest agonists epinephrine and fenoterol provoked 11 to 13 fold increases in the level of $\beta$AR phosphorylation after just 1 min, whereas the weakest agonists dobutamine and ephedrine caused only 3 to 4 fold increases in phosphorylation. With longer treatment times, the level of $\beta$AR phosphorylation declined with the strong agonists, but progressively increased with the weaker partial agonists. The major conclusion drawn from this study is that the occupancy-dependent rate of receptor phosphorylation increases with agonist coupling efficiencies and that this is sufficient to explain the desensitization, internalization, and phosphorylation data obtained.^ The mechanism of activation and desensitization by the partial $\beta$AR agonist salmeterol was also examined in this study. This drug is extremely hydrophobic and its study presents possibly unique problems. To determine whether salmeterol induces desensitization of the $\beta$AR its action has been studied using our system. Employing the use of reversible antagonists it was found that salmeterol, which has an estimated coupling efficiency near that of albuterol caused $\beta$AR desensitization. This desensitization was much reduced relative to epinephrine. Consistent with its coupling efficiency, it was found to be similar to albuterol in its ability to induce internalization and phosphorylation of the $\beta$AR. (Abstract shortened by UMI.) ^

REGULATION OF BRAIN NORADRENERGIC-RECEPTOR FUNCTION BY ADRENOCORTICOTROPHIN AND ANTIDEPRESSANT DRUGS (ADRENERGIC RECEPTOR, CYCLIC-AMP, ADENYLATE CYCLASE, IMIPRAMINE, STRESS)

Human behavior appears to be regulated in part by noradrenergic transmission since antidepressant drugs modify the number and function of (beta)-adrenergic receptors in the central nervous system. Affective illness is also known to be associated with the endocrine system, particularly the hypothalamic-pituitary-adrenal axis. The aim of the present study was to determine whether hormones, in particular adrencorticotrophin (ACTH) and corticosterone, may influence behavior by regulating brain noradrenergic receptor function.^ Chronic treatment with ACTH accelerated the increase or decrease in rat brain (beta)-adrenergic receptor number induced by a lesion of the dorsal noradrenergic bundle or treatment with the antidepressant imipramine. Chronic administration of ACTH alone had no effect on (beta)-receptor number although it reduced norepinephrine stimulated cyclic AMP accumulation in brain slices. Treatment with imipramine also reduced the cyclic AMP response to norepinephrine but was accompanied by a decrease in (beta)-adrenergic receptor number. Both the imipramine and ACTH treatments reduced the affinity of (beta)-adrenergic receptors for norepinephrine, but only the antidepressant modified the potency of the neurotransmitter to stimulate second messenger production. Neither ACTH nor imipramine treatment altered Gpp(NH)p- or fluoride-stimulated adenylate cyclase, cyclic AMP, cyclic GMP, or cyclic GMP-stimulated cyclic AMP phosphodiesterase, or the activity of the guanine nucleotide binding protein (Gs). These findings suggested that post-receptor components of the cyclic nucleotide generating system are not influenced by the hormone or antidepressant. This conclusion was verified by the finding that neither treatment altered adenosine-stimulated cyclic AMP accumulation in brain tissue.^ A detailed examination of the (alpha)- and (beta)-adrenergic receptor components of norepinephrine-stimulated cyclic AMP production revealed that ACTH, but not imipramine, administration reduced the contribution of the (alpha)-receptor mediated response. Like ACTH treatment, corticosterone diminished the (alpha)-adrenergic component indicating that adrenal steroids probably mediate the neurochemical responses to ACTH administration. The data indicate that adrenal steroids and antidepressants decrease noradrenergic receptor function by selectively modifying the (alpha)- and (beta)-receptor components. The functional similarity in the action of the steroid and antidepressants suggests that adrenal hormones normally contribute to the maintenance of receptor systems which regulate affective behavior in man. ^

Opioid dependence treatment: options in pharmacotherapy.

The development of effective treatments for opioid dependence is of great importance given the devastating consequences of the disease. Pharmacotherapies for opioid addiction include opioid agonists, partial agonists, opioid antagonists, and alpha-2-adrenergic agonists, which are targeted toward either detoxification or long-term agonist maintenance. Agonist maintenance therapy is currently the recommended treatment for opioid dependence due to its superior outcomes relative to detoxification. Detoxification protocols have limited long-term efficacy, and patient discomfort remains a significant therapy challenge. Buprenorphine's effectiveness relative to methadone remains a controversy and may be most appropriate for patients in need of low doses of agonist treatment. Buprenorphine appears superior to alpha-2 agonists, however, and office-based treatment with buprenorphine in the USA is gaining support. Studies of sustained-release formulations of naltrexone suggest improved effectiveness for retention and sustained abstinence; however, randomized clinical trials are needed.

Safety, tolerability, and efficacy of fixed combination therapy with dorzolamide hydrochloride 2% and timolol maleate 0.5% in glaucoma and ocular hypertension.

Glaucoma is a collection of diseases characterized by multifactorial progressive changes leading to visual field loss and optic neuropathy most frequently due to elevated intraocular pressure (IOP). The goal of treatment is the lowering of the IOP to prevent additional optic nerve damage. Treatment usually begins with topical pharmacological agents as monotherapy, progresses to combination therapy with agents from up to 4 different classes of IOP-lowering medications, and then proceeds to laser or incisional surgical modalities for refractory cases. The fixed combination therapy with the carbonic anhydrase inhibitor dorzolamide hydrochloride 2% and the beta blocker timolol maleate 0.5% is now available in a generic formulation for the treatment of patients who have not responded sufficiently to monotherapy with beta adrenergic blockers. In pre- and postmarketing clinical studies, the fixed combination dorzolamide-timolol has been shown to be safe and efficacious, and well tolerated by patients. The fixed combination dorzolamide-timolol is convenient for patients, reduces their dosing regimen with the goal of increasing their compliance, reduces the effects of "washout" when instilling multiple drops, and reduces the preservative burden by reducing the number of drops administered per day.

MODELLING β2AR REGULATION

The β2 adrenergic receptor (β2AR) regulates smooth muscle relaxation in the vasculature and airways. Long- and Short-acting β-agonists (LABAs/SABAs) are widely used in treatment of chronic obstructive pulmonary disorder (COPD) and asthma. Despite their widespread clinical use we do not understand well the dominant β2AR regulatory pathways that are stimulated during therapy and bring about tachyphylaxis, which is the loss of drug effects. Thus, an understanding of how the β2AR responds to various β-agonists is crucial to their rational use. Towards that end we have developed deterministic models that explore the mechanism of drug- induced β2AR regulation. These mathematical models can be classified into three classes; (i) Six quantitative models of SABA-induced G protein coupled receptor kinase (GRK)-mediated β2AR regulation; (ii) Three phenomenological models of salmeterol (a LABA)-induced GRK-mediated β2AR regulation; and (iii) One semi-quantitative, unified model of SABA-induced GRK-, protein kinase A (PKA)-, and phosphodiesterase (PDE)-mediated regulation of β2AR signalling. The various models were constrained with all or some of the following experimental data; (i) GRK-mediated β2AR phosphorylation in response to various LABAs/SABAs; (ii) dephosphorylation of the GRK site on the β2AR; (iii) β2AR internalisation; (iv) β2AR recycling; (v) β2AR desensitisation; (vi) β2AR resensitisation; (vii) PKA-mediated β2AR phosphorylation in response to a SABA; and (viii) LABA/SABA induced cAMP profile ± PDE inhibitors. The models of GRK-mediated β2AR regulation show that plasma membrane dephosphorylation and recycling of the phosphorylated β2AR are required to reconcile with the measured dephosphorylation kinetics. We further used a consensus model to predict the consequences of rapid pulsatile agonist stimulation and found that although resensitisation was rapid, the β2AR system retained the memory of prior stimuli and desensitised much more rapidly and strongly in response to subsequent stimuli. This could explain tachyphylaxis of SABAs over repeated use in rescue therapy of asthma patients. The LABA models show that the long action of salmeterol can be explained due to decreased stability of the arrestin/β2AR/salmeterol complex. This could explain long action of β-agonists used in maintenance therapy of asthma patients. Our consensus model of PKA/PDE/GRK-mediated β2AR regulation is being used to identify the dominant β2AR desensitisation pathways under different therapeutic regimens in human airway cells. In summary our models represent a significant advance towards understanding agonist-specific β2AR regulation that will aid in a more rational use of the β2AR agonists in the treatment of asthma.

cAMP-dependent protein kinase and heterologous desensitization of the adenylyl cyclase

Previous experiments had shown no differences in desensitization in cells with mutations of the adenylyl cyclase or the cAMP-dependent protein kinase and had ruled out this kinase as a mediator of desensitization; however, the assays of adenylyl cyclase had been made at high concentrations of free magnesium. The work presented in this dissertation documents a role for cAMP-dependent protein kinase which became apparent with assays at low concentrations of free magnesium. (1) The adenylyl cyclase in membranes from wild type S49 lymphoma cells showed substantial desensitization after incubation of the intact cells with low concentrations of epinephrine (5-20 nM). This desensitization was heterologous, that is it reduced the subsequent responses of the adenylyl cyclase to both epinephrine and prostaglandin-E$\sb1$. (2) The adenylyl cyclase in membranes of S49 cyc$\sp-$ cells, which do not make cAMP in response to hormones, and S49 kin$\sp-$ cells, which lack cAMP-dependent protein kinase activity, showed no heterologous desensitization following incubation of the intact cells with low concentrations of hormones. (3) Heterologous desensitization of the adenylyl cyclase was induced by incubations of wild type cells with forskolin, which activates the adenylyl cyclase downstream of the hormone receptors, or dibutyryl-cAMP, which activates the cAMP-dependent protein kinase directly. (4) Site-directed mutagenesis was used to delete the cAMP-dependent protein kinase consensus phosphorylation sequences on the $\beta$-adrenergic receptor. Heterologous desensitization occurred in intact L-cells expressing the wild type receptor or the receptor lacking the C-terminal phosphorylation site; however, only homologous desensitization occurred when the phosphorylation site on the third intracellular loop of the receptor was deleted. (5) To test directly the effects of cAMP-dependent protein kinase on the adenylyl cyclase the catalytic subunit of the kinase was purified from bovine heart and incubated with adenylyl cyclase in plasma membrane preparations. In this cell-free system the kinase caused rapid heterlogous reductions of the responsiveness of the S49 wild type adenylyl cyclase. Additionally, the adenylyl cyclase in kin$\sp-$ membranes, which showed only homologous desensitization in the intact cell, was desensitization by cell-free incubation with the kinase.^ The epinephrine responsiveness was not affected in L-cell membranes expressing the $\beta$-adrenergic receptor lacking the cAMP-dependent protein kinase consensus sequence on the third intracellular loop. ^

Regulation of adenylyl cyclase by protein kinase C and P$\sb2$ purinergic agonists

Activation of protein kinase C (PKC) causes multiple effects on adenylyl cyclase (AC), (i) an inhibition of (hormone) receptor/G$\sb{\rm s}$ coupling, consistent with PKC modification of the receptor and (ii) a postreceptor sensitization consistent with a PKC-mediated modification of the stimulatory (G$\sb{\rm s}$) or inhibitory (G$\sb{\rm i}$) G-proteins or the catalyst (C) of AC. In L cells expressing the wild-type beta-adrenergic receptor ($\beta$AR) 4-$\beta$ phorbol 12-myristate-13-acetate (PMA) caused 2-3-fold increases in the K$\sb{\rm act}$ and V$\sb{\rm max}$ for epinephrine-stimulated AC activity and an attenuation of GTP-mediated inhibition of AC. Deletion of a concensus site for PKC phosphorylation (amino acids 259-262) from the $\beta$AR eliminated the PMA-induced increase in the K$\sb{\rm act}$, but had no effect on the other actions of PMA. PMA also increased the K$\sb{\rm act}$ and V$\sb{\rm max}$ for prostaglandin E$\sb1$ (PGE$\sb1$)-stimulated AC and the V$\sb{\rm max}$ for forskolin-stimulated AC. Maximal PMA-induced sensitizations were observed when AC was assayed in the presence of 10 $\mu$M GTP and 0.3 mM (Mg$\sp{++}$).^ Liao et al. (J. Biol. Chem. 265:11273-11284 (1990)) have shown that the P$\sb2$ purinergic receptor agonist ATP stimulates hydrolysis of 4,5 inositol bisphosphate (PIP$\sb2$) by phospholipase C (PLC) in L cells. To determine if agonists that stimulate PLC and PMA had similar effects on AC function we compared the effects of ATP and PMA. ATP caused a rapid 50-150% sensitization of PGE$\sb1$-, epinephrine-, and forskolin-stimulated AC activity with an EC$\sb{50}$ of 3 $\mu$M ATP. The sensitization was similar (i.e. Mg$\sp{++}$ and GTP sensitivity) to that caused by 10 nM PMA. However, unlike PMA ATP did not affect the K$\sb{\rm act}$ for hormone-stimulated AC and its effects were unaltered by down-regulation of PKCs following long term PMA treatment. Our results demonstrate that a PKC concensus site in the $\beta$AR, is required for the PMA-induced decrease in receptor/G$\sb{\rm s}$ coupling. Our data also indicate that activation of P$\sb2$ purinergic receptors by ATP may be important in the sensitization of AC in L cells. The mechanism behind this effect remains to be determined. ^

Receptor-mediated adenylylcyclase activation following ``homologous'' and ``heterologous'' desensitization

Using a "collision-coupling" model for $\beta \sb 2$-adrenergic receptor-mediated activation of adenylylcyclase in S49 lymphoma cells, the rate-limiting step of that activation was identified as the association of an "active-state", hormone-bound receptor (HR$\sp\*$) with a G$\sb{\rm s}$-adenylylcyclase moiety (G$\sb{\rm s}$C). It was subsequently hypothesized that the location of the rate-limiting step would not be shifted elsewhere in the activation scheme by receptor desensitization. The traditional focus of receptor desensitization studies has been on modifications of the receptor molecule itself. A "clear-cut" answer to the present hypothesis provides new information on modifications in the function of the receptor following desensitization.^ "Heterologous" desensitization was induced in wild type S49 cells with agents which increase intracellular cAMP without occupying $\beta\sb2$-adrenergic receptors; PGE$\sb1$, forskolin and dibutyryl cAMP. These treatments avoided overlapping effects on $\beta\sb2$-adrenergic receptors by the "homologous" mechanism, in which occupancy by hormone is causative. Although the steady-state activation rate was decreased following heterologous desensitization, that rate was still limited by the association between HR* and G$\sb{\rm s}$C. Thus "heterologous" desensitization acts at the equilibrium between HR and HR* (which is driven by hormone efficiency) such that HR* formation becomes less likely and the frequency of HR*G$\sb{\rm s}$C associations decreases.^ "Homologous" desensitization was induced by high (1-10$\mu$M) epinephrine concentrations in the S49 variant deficient in cAMP-dependent protein kinase, KIN$\sp-$. Use of KIN$\sp-$minimized overlapping effects by the "heterologous" mechanism, which is PKA-dependent. Following homologous desensitization, roughly 50% of the receptors in plasma membrane preparations no longer formed HR*G$\sb{\rm s}$C complexes; evidenced by a decrease in high-affinity hormone binding sites. The loss of HR*G$\sb{\rm s}$C formation did not appear related to the HR/HR* equilibrium. Increasing the efficiency of the assay agonist did nothing to "override" the effect. HR*G$\sb{\rm s}$C association was still the rate-limiting step among the remaining functional receptors. It was not distinguishable whether the remaining activity was "desensitized" due to adenylylcyclase having decreased access to receptors within plasma membrane fragments or due to an effect similar to "heterologous" desensitization. ^

Linkage and association of candidate obesity genes in Mexican-Americans from Starr County, Texas

Obesity and related chronic diseases represent a tremendous public health burden among Mexican Americans, a young and rapidly-expanding population. This study investigated the impact of variation within eight candidate obesity genes, which include leptin (LEP), leptin receptor (LEPR), neuropeptide Y (NPY), NPYY1 receptor (NPYY1), glucagon-like peptide-1 (GLP-1), GLP-1 receptor (GLP1R), beta-3 adrenergic receptor (β3AR), and uncoupling protein (UCP1), on variation in human obesity status and/or quantitative traits related to obesity in Mexican Americans from Starr County, Texas. The Trp64Arg polymorphism within β3AR was typed in 820 random individuals and 240 pedigrees (N = 2,044). The Arg allele frequency was significantly greater in obese versus non-obese individuals (0.20 versus 0. 15, respectively). In addition, within the random sample, the Arg allele was associated with significantly greater body weight (p = 0.031) and body mass index (BMI, p = 0.008) than the Trp allele. In the family sample, the Trp64Arg locus was also linked to percent fat (p = 0.045) but not to body weight or BMI. No linkage between obesity, diabetes, hypertension, or gallbladder disease and the Trp64Arg mutation was observed in families using affected sib pair linkage analysis or the transmission disequilibrium test. Microsatellite markers proximate to the remaining seven genes were typed in 302 individuals from 59 families. Sib pair linkage analysis provided evidence for linkage between obesity and NPY within affected sibling pairs (p = 0.042; n = 170 pairs). NPY was also linked to weight (p = 0.020), abdominal circumference (p = 0.031), hip circumference (p = 0.012), DBP (p ≤ 0.005), and a composite measure of body mass/fat (p ≤ 0.048) in all sibling pairs (n = 545 pairs). Additionally, LEP was linked to waist/hip ratio (p ≤ 0.009), total cholesterol (p ≤ 0.030), and HDL cholesterol (p ≤ 0.026), and LEPR was linked to fasting blood glucose (p ≤ 0.018) and DBP (p ≤ 0.003). Subsequent to the linkage analyses, the NPY gene was sequenced and eight variant sites identified. Two variant sites (-880I/D and 69I/D) were typed in a random sample of 914 individuals. The 880I/D variant was significantly associated with waist/hip ratio (p = 0.035) in the entire sample (N = 914) and with BMI (p = 0. 031), abdominal circumference (p = 0.044), and waist/hip ratio (p = 0.041) in a non-obese subsample (BW < 30 kg/m2, n = 594). The 69I/D variant was a rare mutation observed in only one pedigree and was not associated with obesity or body size/mass within this pedigree. Results of this study indicate that variation at or near β3AR, LEP, LEPR, and NPY may exert effects which increase obesity susceptibility and influence obesity-related measures in this population. ^

STUDIES ON THE PHOSPHORYLATION OF TYROSINE HYDROXYLASE

Tyrosine hydroxylase (E.C. 1.14.16.2, L-tyrosine tetrahydropteridine:oxygen oxidoreductase, 3-hydroxylating), is the initial and rate limiting enzyme in the biosynthetic pathway of catecholamine production. The mechanism by which the activity of tyrosine hydroxylase is altered in response to excitation of adrenergic cells has been suggested to be a covalent modification of the enzyme. A variety of evidence suggests that the stimulus-induced modification of tyrosine hydroxylase responsible for activating the enzyme is an increased phosphorylation of the enzyme. Tyrosine hydroxylase has been shown to be phosphoprotein in situ and undergoes changes in its state of phosphorylation upon stimulation of the adrenergic tissue. Further, in vitro phosphorylation of tyrosine hydroxylase increases the activity of the enzyme in a manner kinetically similar to the changes observed in the enzyme after stimulation of the intact adrenergic tissue. Thus, the covalent modification of tyrosine hydroxylase by reversible phosphorylation appears to provide a rapid and sensitive mechanism of coupling the activity of the enzyme to the excitation process. The mechanism by which the adrenergic cell mediates the depolarization-dependent phosphorylation and activation of tyrosine hydroxylase is controversial. The most accepted working model suggests that the cAMP-dependent protein kinase mediates this process, however a variety of data are inconsistent with this hypothesis.^ This dissertation attempts to identify the protein kinase(s) responsible for mediating the stimulus-dependent phosphorylation of tyrosine hydroxylase in purified, isolated bovine adrenal chromaffin cells. These studies address this question by first identifying the protein kinase activities in the chromaffin cells which can phosphorylate tyrosine hydroxylase and subsequently, evaluating the possibility that these protein kinases mediate the stimulus-dependent phosphorylation of the enzyme by tryptic peptide mapping. The maps of tyrosine hydroxylase phosphorylated by these protein kinase activities were compared with that of tyrosine hydroxylase phosphorylated in situ. The outcome of these studies have been the identification of three protein kinase activities in the chromaffin cells which can phosphorylate tyrosine hydroxylase in vitro, and the determination that one, a calcium-, calmodulin-dependent protein kinase, is capable of accounting for the pattern of phosphate incorporation into tyrosine hydroxylase observed in situ. The results of these experiments suggest that the depolarization-dependent activation of tyrosine hydroxylase in adrenal chromaffin cells may be mediated by the activation of a calcium-, calmodulin-dependent protein kinase by the influx of calcium into the cells and the subsequent phosphorylation of tyrosine hydroxylase by this enzyme.^

The role of interleukin-6 in the angiogenesis of ovarian carcinoma

The purpose of this study was to characterize the effects of IL-6 on endothelial cells and to investigate the role of IL-6 in the angiogenesis of ovarian carcinomas. We evaluated human ovarian carcinoma clinical specimens and determined that high expression of IL-6 was associated with increased tumor vascularization. Additionally, endothelial cells derived from the ovary and mesentery expressed the IL-6 receptor (IL-6R), and their stimulation with the exogenous ligand activated downstream signaling molecules and enhanced cell migration. Dual immunohistochemical staining for CD-31 and IL-6R revealed IL-6R expression on human endothelial cells within normal ovary and ovarian carcinomas. To further investigate the possible proangiogenic function of IL-6, Gelfoam sponges containing IL-6 or bFGF were implanted into the subcutis of BALB/c mice. IL-6 containing sponges were vascularized to the same extent as bFGF containing sponges. ^ Chronic stress can adversely affect disease progression. Stimulation of ovarian carcinoma cell lines with concentrations of catecholamines achieved in individuals experiencing chronic stress resulted in a substantial increase in IL-6 production. It was determined that stress mediators regulate IL-6 expression through the β-adrenergic receptor and Src. These data illustrate one mechanism by which chronic stress may influence tumor progression. ^ To investigate whether IL-6 contributes to the angiogenesis of ovarian carcinomas, we isolated low IL-6 expressing clones from the SKOV3.ip1 cell line and transfected them with a plasmid encoding the IL-6 gene. We observed no difference in tumor weight between high and low IL-6 expressing cells. However, while low IL-6 expressing tumors were highly vascularized, high IL-6 expressing tumors appeared hypervascularized. Immunohistochemical analysis revealed that all tumors exhibited robust expression of additional proangiogenic molecules. ^ Collectively, these studies indicate that IL-6 secreted by ovarian cancer cells is a highly proangiogenic cytokine. However, IL-6 is but one of several proangiogenic molecules produced by ovarian cancer, and its inhibition may not be sufficient to inhibit angiogenesis of ovarian carcinoma. The findings presented in this dissertation provide insight into the function of IL-6 as a regulator of angiogenesis. Understanding of the role of proangiogenic molecules such as IL-6 in ovarian carcinoma may have important implications for therapy directed at the vascular component of this disease. ^