<p>Conclusion of this study: These data suggest that 6 weeks of high-fat feeding induces hypertension but does not produce obesity, dyslipidemia and insulin resistance. However, this model may be useful in studying vascular reactivity in hypertension in the absence of insulin resistance.</p>

Intratympanic injection of various otoprotectants through the round window membrane (RWM) might become available in the near future as an alternative to the currently available medical and surgical methods used to treat several inner ear diseases. The most common outcome of such diseases is sensorineural hearing loss (SNHL). Two examples of  these otoprotectants are Edaravone and Brain-Derived Neurotrophic Factor (BDNF), both of which have already proved effective against  noise-induced hair cell loss, barotrauma  and ototoxicity caused by cisplatin. In four different studies we used two electrophysiological methods, auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE), to study the effects of tobramycin and Pseudomonas aeruginosa exotoxin A (PaExoA) on the inner ears of 129 male Sprague-Dawley rats. In two investigations, not only the otoprotective effects of Edaravone on tobramycin-induced ABR threshold shifts and PaExoA-induced DPOAE  threshold changes, were studied but even different application times, in order to establish in which interval it was still possible to achieve effective otoprotection.We found that Edaravone gave otoprotection from tobramycin when injected simultaneously or within 7 days, but it had only a limited effect on the changes in DPOAE thresholds caused by PaExoA when injected 1, 2, or 4 hours after the exotoxin. The effect of BDNF on PaExoA-induced ABR threshold shifts was investigated in two studies, where different doses of intratympanically injected PaExoA were used and where BDNF was applied simultaneously, 12 or 72 hours efter exotoxin instillation. We found that BDNF had an otoprotective effect on SNHL induced by different doses PaExoA when injected simultaneously or with no more than 12 hours delay.

Background and Purpose: The endothelium is a single-cell layer positioned at the blood-vascular wall interface, where in response to blood-borne signals and hemodynamic forces, endothelial cells act as central regulators of vascular homeostatic processes including vascular tone, growth and remodeling, inflammation and adhesion, and blood fluidity and coagulation. Agonist- or flow-stimulated endothelium-dependent vasorelaxation becomes impaired in states of cardiovascular disease (CVD) risk and has been identified as a possible biomarker of overall endothelial dysfunction leading to vascular dysregulation and disease pathogenesis. Accordingly, it is important to elucidate the mechanisms accounting for this endothelial vasomotor dysfunction. Upon stimulation, endothelial cells can synthesize and release a variety of endothelium-derived relaxing factors (EDRFs), the most prominent of which is nitric oxide (NO) derived from NO synthase (NOS). In addition, under certain CVD risk conditions including hypertension and aging, stimulated endothelial cells can become a prominent source of endothelium-derived contracting factors (EDCFs) produced in a cyclooxygenase (COX)-dependent manner. Consequently, endothelial dysfunction may be caused by under-active EDRF signaling and/or competitive over-active EDCF signaling. Much attention has been given to elucidating the mechanisms of under-active EDRF signaling and its role in causing endothelial dysfunction, wherein excess reactive oxygen species (ROS) accumulation and oxidative stress under CVD risk conditions have been recognized as major factors in reducing NO bioavailability thus causing under-active EDRF signaling and endothelial dysfunction. Less attention however, has been given to elucidating the mechanisms of over-active COX-mediated EDCF signaling and its role in causing endothelial dysfunction. Moreover, while COX-mediated EDCF signaling activity has been investigated in some segments of the vasculature, most notably the aorta, it has not been well-investigated in the common carotid artery (CCA), a highly accessible cerebral blood flow conduit particularly advantageous in exploring the roles of the endothelium in vascular pathogenesis. It was the global purpose of this thesis to gain a better understanding of the cellular-molecular mechanisms accounting for endothelial dysfunction in the CCA of animal models known to exhibit COX-mediated EDCF signaling activity, in particular essential (spontaneous) hypertension and aging. Experimental Objective and Approach: This thesis comprises three studies. Study I and Study II investigated the CCA of young-adult (16-24wk old) normotensive Wistar Kyoto (WKY) and Spontaneously Hypertensive (SHR) rats. Study III investigated the CCA of Adult (25-36wks old) and Aging (60-75wks old) Sprague Dawley (SD) rats treated in vivo (or not; CON) with L-buthionine sulfoximine (BSO) to chronically deplete the cellular anti-oxidant glutathione (GSH) and increase ROS accumulation and oxidative stress. The global objective and approach across these studies was to systematically examine the relative contributions of NOS and COX signaling pathways in mediating the acetylcholine (ACh)-stimulated endothelium-dependent relaxation (EDRF) and contractile (EDCF) activities of isometrically-mounted CCA in tissue baths in vitro, with a particular focus on elucidating the mechanisms of COX-mediated EDCF signaling activity. An added objective was to examine the in vivo hemodynamic characteristics of the CCA in each animal model investigated, serving both to identify the pressure-flow environment that the CCA is exposed to in vivo and to provide assessment of potential hypertension, aging, and oxidative stress effects on large artery hemodynamics. Key Findings: Study I hemodynamic analysis confirmed a hypertensive state in young adult SHR while also exposing a reduction in mean CCA blood flow in SHR compared to WKY accompanied by a multi-faceted pressure-flow interaction across the cardiac cycle relating to flow and pressure augmentation. Study III hemodynamic analysis found that neither aging nor chronic BSO-induced GSH depletion affected CCA blood pressure or blood flow parameters in SD rats. Study I and II demonstrated that a COX-mediated EDCF response impaired ACh-stimulated endothelium-dependent vasorelaxation in pre-contracted CCA from young adult SHR, while EDRF signaling activity, predominantly mediated by NO, remained well-preserved compared to WKY. Examining ACh-stimulated contractile function specifically from a quiescent (non pre-contracted) state revealed that EDCF activity did exist in WKY CCA but could be completely suppressed by NO-mediated EDRF signaling activity, whereas the similarly robust NO-meditated EDRF signaling activity in SHR CCA could not fully suppress its >2-fold augmented EDCF activity vs. WKY CCA. Further pharmaco-dissection of ACh-stimulated contractile function in the SHR-WKY CCA model revealed that the EDCF signaling activity was completely dependent on the COX-1 (but not COX-2) isoform of COX and was almost exclusively mediated by the thromboxane-prostanoid (TP) sub-type of the prostaglandin (PG) G-protein coupled receptor family and by Rho-associated kinase (ROCK), a down-stream effector of the molecular switch RhoA. Furthermore, it was found that while exogenous ROS-stimulated CCA contractile function was similarly >2-fold augmented in SHR vs. WKY and dependent on COX-1 and TP receptor and ROCK effectors, ACh-stimulated CCA EDCF signaling activity was only minimally affected by in-bath ROS manipulating compounds. Additional biochemical and molecular analysis revealed that ACh stimulation was associated with PG over-production from an over-expressed COX-1 in SHR CCA, and with CCA plasma membrane localization and activation of RhoA. Study III demonstrated that a COX-mediated EDCF response impaired ACh-stimulated endothelium-dependent vasorelaxation in pre-contracted CCA from Aging SD rats, while EDRF signaling activity, predominantly mediated by NO, remained well-preserved compared to Adult SD rats. Specific examination of ACh-stimulated contractile function revealed that EDCF activity did exist in Adult CCA but could be completely suppressed by NO-mediated EDRF signaling activity, whereas the similarly robust NO-meditated EDRF signaling activity in Aging CCA could not fully suppress its >3-fold augmented EDCF activity vs. Adult CCA. Further pharmaco-dissection of ACh-stimulated contractile function in the Adult-Aging SD rat CCA model revealed that EDCF signaling activity was completely dependent on COX-1, but while exogenous ROS was able to elicit a COX-dependent CCA contractile response, in-bath ROS manipulating compounds were found to be without effect on ACh-stimulated CCA EDCF signaling activity. Furthermore, biochemical analysis revealed that aging was not associated with a change in tissue (liver and vascular) GSH content or ROS accumulation. Chronic in vivo BSO treatment was effective in depleting tissue GSH content and increasing ROS accumulation, to a similar extent, in both Adult and Aging SD rats. However, regardless of age, neither ACh-stimulated NO-mediated EDRF signaling activity nor COX-mediated EDCF signaling activity were affected by these BSO-induced perturbations. Conclusions and Perspective: In the CCA of animals at the early pathological stages of either essential hypertension (young adult SHR) or normotensive aging (Aging SD rats), endothelial vasomotor dysfunction can be caused solely by over-active EDCF signaling, apparently disconnected from changes in NO bioavailability or oxidative stress. While NO and ROS may act, respectively, as negative and positive modulators of the established COX-PG-TP receptor-RhoA-ROCK cell-signaling axis mediating endothelium-dependent contractile activity, these factors do not appear to be essential to the mechanism(s) underlying the development of over-active EDCF signaling. Further elucidation of the cellular-molecular causes of over-active EDCF signaling, and its patho-biological consequences, in the SHR-WKY and Adult-Aging SD rat CCA models of EDCF activity established and hemodynamically characterized in this thesis, may help to identify new or more effective targets to be used in prevention or treatment strategies to combat the pathogenesis of CVD.

Long-term potentiation (LTP) and depression (LTD) are important forms of activity-dependent synaptic plasticity believed to play a role in memory at the cellular level. It has previously been described that synthesis of new proteins is needed to maintain LTP longer than a few hours. Other reports argue that sufficient proteins for stable LTP are already available. The present study aims to examine the role of protein synthesis in LTD, the presumed mirror mechanism of LTP. Experiments were carried out in hippocampal slices from young (12-45 days) and old (12-18 weeks) Sprague-Dawley rats. Extracellular techniques were used to study synaptic responses in the Schaffer-collateral-commissural pathway. Plasticity was induced electrically by low frequency stimulation (2-3 trains at 1 Hz for 15 min) or chemically by brief exposure to certain glutamate receptor agonists (NMDA at 20 µM for 3 min or DHPG at 100 µM for 10 min). Whole slice protein synthesis was quantified by assessing 3H-leucine incorporation. Stable LTD (&gt; 8 h) was be obtained by either electrical or chemical activation. Protein synthesis inhibitors anisomycin (40 uM) and cycloheximide (100 uM) both failed to influence the magnitude of LTD. Moreover, no age difference was found, in terms of stable LTD in both young and old rats under inhibition of protein synthesis. The potency of the inhibitors was found to be high, depressing synthesis down to a few percent. It is concluded that sufficient proteins for generating stable LTD are normally present in the brain, implying a large safety-margin for cellular memory.

Cerebral ischemia is the principal cause of morbidity and mortality worldwide. In addition to neuronal loss associated with hypoxic-ischemic damage, cerebral ischemia is characterized by a neuromicrovascular injury. Nitrative stress and lipid peroxidation increase in hypoxic-ischemic damages and play an essential role in neuromicrovascular injury leading to cerebral ischemia. We hypothesized that newly described lipid peroxidation products, termed trans-arachidonic acids (TAA), could be implicated in the pathogenesis of hypoxia-ischemia by affecting the cerebral vasomotricity and microvascular integrity. / The effects of TAA on neuromicrovascular tone were tested ex vivo by monitoring the changes in vascular diameter of rat cerebral pial microvessels. Four isomers of TAA, namely 5 E-AA, 8E-AA, IIE-AA and 14 E-AA induced an endothelium-dependent vasorelaxation. Possible mechanisms involved in TAA-induced vasorelaxation were thoroughly investigated. Collectively, data enclosed revealed that TAA induce cerebral vasorelaxation through the interactive activation of BKCa channels with heme oxygenase-2. This interaction leads to generation of carbon monoxide which in turn activates soluble guanylate cyclase and triggers vasorelaxation. / Chronic effects of TAA on microvascular integrity were examined by generating a unilateral hypoxic-ischemic (HI) model of cerebral ischemia on newborn rat pups. Our HI model showed microvascular degeneration as early as 24h post-HI, preceded by an increase in cerebral TAA levels. HI-induced microvascular lesions were dependent on nitric oxide synthase activation and ensued TAA formation. Although the molecular mechanisms leading to TAA-induced microvascular degeneration were, in part uncovered for the retina, the primary site of action of TAA remains unknown. We demonstrated that TAA binds and activates GPR40 receptor, a newly described free fatty acid receptor. Importantly, GPR40 receptor knock-out prevents TAA-induced reduction in cerebral microvascular density and limits HI-induced brain infarct.

Hypertension is the number one cause of death worldwide. Low-grade inflammation has been identified as one of the mechanisms contributing to blood pressure elevation and remodeling of the vasculature in hypertension. Mechanisms involved in vascular inflammation and hypertension remain elusive. Vasoactive peptides such as endothelin-1 (ET-1) and angiotensin II (Ang II), oxidative stress and infiltration of immune cells are increased in cardiovascular tissues of hypertensive individuals. Since the vasculature is a major regulator of blood pressure levels, the hypothesis has been proposed that vascular inflammatory responses contribute to development of hypertension. / Objectives of this thesis were 1) to investigate the role of T cells in development of vascular inflammation observed in genetically hypertensive rats, 2) to identify vascular sources of reactive oxygen species production in mineralocorticoid-induced hypertension and 3) to study the effect of peroxisome proliferator-activated receptor (PPAR)-gamma activators on vascular pro-inflammatory signaling pathways in Ang II-induced hypertension. / The first study that is part of this thesis shows that the transfer of chromosome 2 from normotensive to hypertensive rats reduces plasma levels of pro-inflammatory cytokines, expression of adhesion molecules and infiltration of T cells in aorta as well as resulting in lower blood pressure levels. These effects are accompanied by increased regulatory T cell mediators. We discovered that regulatory T cells are regulated by chromosome 2 and may be responsible for reducing inflammatory responses in hypertensive rats. / The second study of this thesis demonstrates in DOCA-salt hypertensive rats that superoxide (&middot;O2-) production originates in part from xanthine oxidase activity induced by the ET-1 system and from mitochondrial sources, particularly complex II of the respiratory chain. We thus have uncovered two sources of reactive oxygen species (ROS) that can stimulate inflammatory responses in hypertension, since vascular &middot;O 2- production in this model was shown to induce vascular inflammation. / The third study of the thesis shows that activators of PPAR-gamma reduce blood pressure levels and signaling pathways including Akt/PKB, SHIP2, ERK1/2, 4E-BP1 in aorta and resistance arteries in Ang II-induced hypertension. PPARy acts as an anti-inflammatory transcription factor, and the present study suggests that Ang II down-regulates PPAR-gamma activity to exert its pro-inflammatory effects. / In conclusion, by targeting inflammatory mediators, it may be possible to reduce blood pressure levels in hypertensive animals. This suggests that inflammatory responses may play a crucial role in development of high blood pressure.

<p>Conclusion of this study: These data suggest that 6 weeks of high-fat feeding induces hypertension but does not produce obesity, dyslipidemia and insulin resistance. However, this model may be useful in studying vascular reactivity in hypertension in the absence of insulin resistance.</p>

Background and Purpose: The endothelium is a single-cell layer positioned at the blood-vascular wall interface, where in response to blood-borne signals and hemodynamic forces, endothelial cells act as central regulators of vascular homeostatic processes including vascular tone, growth and remodeling, inflammation and adhesion, and blood fluidity and coagulation. Agonist- or flow-stimulated endothelium-dependent vasorelaxation becomes impaired in states of cardiovascular disease (CVD) risk and has been identified as a possible biomarker of overall endothelial dysfunction leading to vascular dysregulation and disease pathogenesis. Accordingly, it is important to elucidate the mechanisms accounting for this endothelial vasomotor dysfunction. Upon stimulation, endothelial cells can synthesize and release a variety of endothelium-derived relaxing factors (EDRFs), the most prominent of which is nitric oxide (NO) derived from NO synthase (NOS). In addition, under certain CVD risk conditions including hypertension and aging, stimulated endothelial cells can become a prominent source of endothelium-derived contracting factors (EDCFs) produced in a cyclooxygenase (COX)-dependent manner. Consequently, endothelial dysfunction may be caused by under-active EDRF signaling and/or competitive over-active EDCF signaling. Much attention has been given to elucidating the mechanisms of under-active EDRF signaling and its role in causing endothelial dysfunction, wherein excess reactive oxygen species (ROS) accumulation and oxidative stress under CVD risk conditions have been recognized as major factors in reducing NO bioavailability thus causing under-active EDRF signaling and endothelial dysfunction. Less attention however, has been given to elucidating the mechanisms of over-active COX-mediated EDCF signaling and its role in causing endothelial dysfunction. Moreover, while COX-mediated EDCF signaling activity has been investigated in some segments of the vasculature, most notably the aorta, it has not been well-investigated in the common carotid artery (CCA), a highly accessible cerebral blood flow conduit particularly advantageous in exploring the roles of the endothelium in vascular pathogenesis. It was the global purpose of this thesis to gain a better understanding of the cellular-molecular mechanisms accounting for endothelial dysfunction in the CCA of animal models known to exhibit COX-mediated EDCF signaling activity, in particular essential (spontaneous) hypertension and aging. Experimental Objective and Approach: This thesis comprises three studies. Study I and Study II investigated the CCA of young-adult (16-24wk old) normotensive Wistar Kyoto (WKY) and Spontaneously Hypertensive (SHR) rats. Study III investigated the CCA of Adult (25-36wks old) and Aging (60-75wks old) Sprague Dawley (SD) rats treated in vivo (or not; CON) with L-buthionine sulfoximine (BSO) to chronically deplete the cellular anti-oxidant glutathione (GSH) and increase ROS accumulation and oxidative stress. The global objective and approach across these studies was to systematically examine the relative contributions of NOS and COX signaling pathways in mediating the acetylcholine (ACh)-stimulated endothelium-dependent relaxation (EDRF) and contractile (EDCF) activities of isometrically-mounted CCA in tissue baths in vitro, with a particular focus on elucidating the mechanisms of COX-mediated EDCF signaling activity. An added objective was to examine the in vivo hemodynamic characteristics of the CCA in each animal model investigated, serving both to identify the pressure-flow environment that the CCA is exposed to in vivo and to provide assessment of potential hypertension, aging, and oxidative stress effects on large artery hemodynamics. Key Findings: Study I hemodynamic analysis confirmed a hypertensive state in young adult SHR while also exposing a reduction in mean CCA blood flow in SHR compared to WKY accompanied by a multi-faceted pressure-flow interaction across the cardiac cycle relating to flow and pressure augmentation. Study III hemodynamic analysis found that neither aging nor chronic BSO-induced GSH depletion affected CCA blood pressure or blood flow parameters in SD rats. Study I and II demonstrated that a COX-mediated EDCF response impaired ACh-stimulated endothelium-dependent vasorelaxation in pre-contracted CCA from young adult SHR, while EDRF signaling activity, predominantly mediated by NO, remained well-preserved compared to WKY. Examining ACh-stimulated contractile function specifically from a quiescent (non pre-contracted) state revealed that EDCF activity did exist in WKY CCA but could be completely suppressed by NO-mediated EDRF signaling activity, whereas the similarly robust NO-meditated EDRF signaling activity in SHR CCA could not fully suppress its >2-fold augmented EDCF activity vs. WKY CCA. Further pharmaco-dissection of ACh-stimulated contractile function in the SHR-WKY CCA model revealed that the EDCF signaling activity was completely dependent on the COX-1 (but not COX-2) isoform of COX and was almost exclusively mediated by the thromboxane-prostanoid (TP) sub-type of the prostaglandin (PG) G-protein coupled receptor family and by Rho-associated kinase (ROCK), a down-stream effector of the molecular switch RhoA. Furthermore, it was found that while exogenous ROS-stimulated CCA contractile function was similarly >2-fold augmented in SHR vs. WKY and dependent on COX-1 and TP receptor and ROCK effectors, ACh-stimulated CCA EDCF signaling activity was only minimally affected by in-bath ROS manipulating compounds. Additional biochemical and molecular analysis revealed that ACh stimulation was associated with PG over-production from an over-expressed COX-1 in SHR CCA, and with CCA plasma membrane localization and activation of RhoA. Study III demonstrated that a COX-mediated EDCF response impaired ACh-stimulated endothelium-dependent vasorelaxation in pre-contracted CCA from Aging SD rats, while EDRF signaling activity, predominantly mediated by NO, remained well-preserved compared to Adult SD rats. Specific examination of ACh-stimulated contractile function revealed that EDCF activity did exist in Adult CCA but could be completely suppressed by NO-mediated EDRF signaling activity, whereas the similarly robust NO-meditated EDRF signaling activity in Aging CCA could not fully suppress its >3-fold augmented EDCF activity vs. Adult CCA. Further pharmaco-dissection of ACh-stimulated contractile function in the Adult-Aging SD rat CCA model revealed that EDCF signaling activity was completely dependent on COX-1, but while exogenous ROS was able to elicit a COX-dependent CCA contractile response, in-bath ROS manipulating compounds were found to be without effect on ACh-stimulated CCA EDCF signaling activity. Furthermore, biochemical analysis revealed that aging was not associated with a change in tissue (liver and vascular) GSH content or ROS accumulation. Chronic in vivo BSO treatment was effective in depleting tissue GSH content and increasing ROS accumulation, to a similar extent, in both Adult and Aging SD rats. However, regardless of age, neither ACh-stimulated NO-mediated EDRF signaling activity nor COX-mediated EDCF signaling activity were affected by these BSO-induced perturbations. Conclusions and Perspective: In the CCA of animals at the early pathological stages of either essential hypertension (young adult SHR) or normotensive aging (Aging SD rats), endothelial vasomotor dysfunction can be caused solely by over-active EDCF signaling, apparently disconnected from changes in NO bioavailability or oxidative stress. While NO and ROS may act, respectively, as negative and positive modulators of the established COX-PG-TP receptor-RhoA-ROCK cell-signaling axis mediating endothelium-dependent contractile activity, these factors do not appear to be essential to the mechanism(s) underlying the development of over-active EDCF signaling. Further elucidation of the cellular-molecular causes of over-active EDCF signaling, and its patho-biological consequences, in the SHR-WKY and Adult-Aging SD rat CCA models of EDCF activity established and hemodynamically characterized in this thesis, may help to identify new or more effective targets to be used in prevention or treatment strategies to combat the pathogenesis of CVD.

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 214 pages. Includes vita. Includes bibliographical references.

The purpose of this investigation was to examine selected biochemical mechanisms known to influence contractility and energy metabolism in the myocardium, with particular emphasis placed on the regulatory role of protein phosphorylation in the ventricular myocardium. The investigation was conducted in three phases; initially the cardiac contraction cycle was examined to determine whether reported fluctuations in myocardial cAMP levels were associated with other biochemical events known to be cAMP-dependent. The second phase involved the determination of specific kinase activities and endogenous substrates in a highly purified cardiac sarcolemmal preparation. In the final phase, ventricular myocytes were utilized to examine the ability of adenosinergic and muscarinic agonists to influence the isoproterenol-induced increases in protein phosphorylation. Studies in the first phase examined cyclic AMP levels and selected kinase activities in hearts frozen at various stages of the cardiac cycle. An automated clamping device, capable of freezing a perfused rat heart in less than 50 msec, was utilized to separate the cardiac cycle into various phases. Three different timing schemes were employed to divide the cycle into 2 to 4 segments. These different timing schemes revealed no significant differences in cAMP during the cardiac cycle. Myocardial cAMP values ranged from 2.5 to 4.1 pmol/min/mg protein in all phases. However, in one scheme there was a tendency for cAMP to be elevated in early systole, with minimal values occurring diastole. There were also no significant differences seen for either glycogen phosphorylase or cAMP-dependent protein kinase (PKA) activity between various phases of the cardiac cycle. Since no significant fluctuations were observed in the levels of cAMP or the activities of PKA or glycogen phosphorylase during a single cardiac contraction cycle, it would appear that these agents do not exert their effects on cardiac function on a beat to beat basis. The second phase of study examined the nature and function of individual protein kinases in the myocardium. Using a highly purified cardiac sarcolemmal preparation, kinase specific, synthetic substrates were employed to quantify the activities of cAMP-dependent (PKA), calcium/calmodulin-dependent (PKCM), calcium/phospholipid-dependent (PKC) and cGMP-dependent (PKG) protein kinases. Additionally, endogenous protein substrates were examined in this preparation to provide possible insight as to the function of these kinases in the heart. The activities of PKA, PKG, PKCM, and PKC in nmol 32P/min/μg protein were as follows: PKA, 1606; PKG, 35.7; PKCM, 353; and PKC, 13.2. Three endogenous protein substrates of apparent molecular weights of 15kD, 28kD and 92kD were phosphorylated. While no endogenous protein phosphorylation was detectable as a result of cG-PK activity, all of the substrates were phosphorylated, to varying degrees, by both PKA and CACM-PK. PKC phosphorylated only the 15kD substrate. Even though several endogenous kinases are evident in the sarcolemmal preparation, cAMP-dependent protein kinase demonstrates the greatest degree of activity. This kinase also appeared to be the most abundant; however, there is some concern as to the source of these kinases in the membrane preparation since endothelial membranes as well as cardiac membranes appeared to be present. Evidence for endothelial contamination was provided by the finding that the membrane preparation contained appreciable amounts of angiotensin converting enzyme (ACE) activity, an enzyme felt to reside in the vascular endothelium. Since studies with this preparation could not exclude contribution of nonmuscle cell membranes a model consisting solely of dispersed ventricular myocytes was developed. The third phase of these studies examined protein phosphorylation in primary cultures of ventricular myocytes. Specifically, these studies examined protein phosphorylation induced by exposure to isoproterenol (ISO), a catecholamine known to effect changes in the phosphorylation state of proteins in the heart by means of a β-adrenergic-mediated/cAMP-dependent mechanism was examined. Additionally, the effects of phenylisopropy-ladenosine (PIA) and carbamyl choline chloride (CARB) were examined with regard to their anti-adrenergic role(s) in this process. Adherent, collagenase-dispersed, radiolabelled (32p) ventricular myocytes exposed to ISO demonstrated a dose and time dependent increase in 32p incorporation into several endogenous protein substrates. When the myocytes were exposed (60 sec) to either PIA or CARB prior to the exposure to ISO, ISO-induced 32p incorporation into protein substrates of apparent molecular weight of 6kD, 31kD and 155kD was reduced up to 67% when compared to the effects of ISO alone. Additionally, both PIA and CARB attenuated the ISO-induced increase in PKA activity in the myocyte, yet only CARB was seen to produce an inhibitory effect on the ISO-induced increase in cAMP levels in the myocytes. The effects of CARB were dose-dependent and inhibited the effects of ISO on 32p incorporation at all doses tested. PIA elicited biphasic effects: lower PIA concentrations were inhibitory in nature, while higher concentrations of PIA appeared to potentiate the increase in 32p incorporation induced by ISO. Based on electrophoretic mobilities (SDS/PAGE) of the 6kD and the 155kD substrates, these substrates have been tentatively identified as the monomeric form of the sarcoplasmic reticulum-associated protein, phospholamban, and the contractile filament-associated protein, C protein, respectively. The 31kD substrate has been identified, by means of immunoblot, as the contractile filament-associated protein, troponin I. The role of protein phosphorylation in the myocardium involves complex, inter-related mechanisms that encompass extracellular, transmembranal and cytoplasmic elements in the heart. It is well understood that certain mechanisms of the contraction cycle known to vary on a beat to beat basis, such as myosin ATPase, involve changes in protein phosphorylation. However, the nature of the various kinases and substrates examined in this study appear to influence longer-term events of myocardial contractility. Mechanisms coupled with hormone action, modulation of second messenger-dependent components, and factors associated with changes in contractility seen with aging and disease are more likely to exhibit changes similar to those described herein. A better understanding of the underlying biochemistry may provide greater insight into the importance of these metabolic changes.