Comparison of energy-minimized structures of [Pd(II)(N-methyliminodiacetate)] complexes of X(1)-His-X(3)-His-His peptides as an analysis of steric and specific interactions with synthetic binding tags for IMAC separations.

[Pd(II)(mida)(peptide)] complexes for the series of peptides of sequence X(1)-His-X(3)-His-His were studied by molecular mechanics methods using Spartan, MMFF94, and SYBYL programs with X(1) = X(3) = glycine (G), phenylalanine (F), tyrosine (Y), tryptophan (W), and with X(1) = glycine (G) and X(3) = proline (P). For comparison purposes, data were also obtained for the Ser-Pro-His-His-Gly (SPHHG) and the (His)(5) peptides.The latter two peptides and GHPHH are tags in current use for IMAC separations. These provide calibration points as to the binding affinities that have been determined for the entire series. The energies of the complexes, as an average trend found from the composite behavior of the three methods, were found to be SPHHG (205 kcal/mol) (most stable; are values obtained by MMFF94 methods) < HH(#)HH(#)H(#) (222; where # implies the site of attachment to match the other X(1)-His-X(3)-His-His peptides) < YHYHH (249) < GHGHH (265) < WHWHH (284) approximately GHPHH (286) < FHFHH (311) (least stable), implying that FHFHH might be a useful chromatographic tag for IMAC protein separations that would elute more readily than GHPHH from IMAC sites that are of square-planar structure, such as Cu(II)(ida-supported) IMAC columns. Specific H-bonded interactions are observed between the tyrosine X(1) and pendant carboxylates and between X(3) and the N-terminal amine of [Pd(mida)(YHYHH)]. Face-to-pi-face ring stacking occurs between phenylalanine X(1) and X(3) units in [Pd(mida)(FHFHH)], whereas edge C-H to pi H-bonding or pi stacking occurs between the X(1) and X(3) tryptophans of [Pd(mida)(WHWHH)]. Two energy minima were found with tryptophan. The more stable form has the aromatic rings more parallel, similar to the stacked form of phenylalanine, rather than the edge C-H to pi H-bonding, and virtually the same overall energy as for [Pd(mida)(GHPHH)]. The "perpendicular" structure was found as an initial local energy minimum, but additional MMFF94 calculations found the pi -stacked arrangement at energy ca. 39 kcal/mol lower than that of the nearly "perpendicular" arrangement of the tryptophan rings, a composite effect of relaxation of the peptide, together with differences in stabilities imparted by the differing geometries. The use of the terms "pi-stacked " and "perpendicular" forms represent the limiting cases available to the tryptophan side chain groups. A twist of about 15 degrees to 20 degrees in dihedral angle is all that is necessary to change between structures that are nearly described as one form or the other.

The effects of 17 alpha-methyltestosterone on myocardial function in vitro.

Testosterone analogs have been used as performance enhancers by athletes for more than 40 yr. We asked whether the anabolic steroid 17 alpha-methyl-4-androstene-17-ol-3-one (17 alpha-MT) would affect intrinsic contractile function of the heart. Male Sprague-Dawley rats, 125-150 g, were treated with 17 alpha-MT either parenterally or orally for up to 8 wk. Intrinsic contractile function of the hearts was assessed utilizing both the isolated working heart and isovolumic perfused heart preparations. Isolated working hearts from 17 alpha-MT-treated rats had a 45% decrease in heart work attributable largely to a similarly decreased stroke volume. Isovolumic perfused hearts from treated animals had elevated left ventricular systolic and diastolic pressures at similar interventricular volumes compared to controls. Rates of ventricular pressure development (+dP/dT) or relaxation (-dP/dT) were unchanged as a result of the treatment. However, static elastance was reduced in potassium-arrested hearts from the 17 alpha-MT treatment (63% increase in interventricular pressure), consistent with a limitation being imposed on stroke volume by a decreased myocardial compliance. Hydroxyproline content of the hearts was not altered by 17 alpha-MT treatment suggesting that increased stiffness was not a consequence of collagen proliferation. Treatment of the steroid rats with beta-aminopropionitrile, a compound that inhibits lysyl oxidase, restored the left ventricular volume-pressure relationship (elastance curve) to that of control hearts. Thus, chronic treatment with anabolic steroids appears to reduce left ventricular compliance, possibly related to an enhanced activity of lysyl oxidase, and results in increased crosslink formation between collagen strands in the extracellular matrix.

Design of affinity tags for one-step protein purification from immobilized zinc columns.

Affinity tags are often used to accomplish recombinant protein purification using immobilized metal affinity chromatography. Success of the tag depends on the chelated metal used and the elution profile of the host cell proteins. Zn(II)-iminodiacetic acid (Zn(II)-IDA) may prove to be superior to either immobilized copper or nickel as a result of its relatively low binding affinity for cellular proteins. For example, almost all Escherichia coli proteins elute from Zn(II)-IDA columns between pH 7.5 and 7.0 with very little cellular protein emerging at pH values lower than 7.0. Thus, a large portion of the Zn(II)-IDA elution profile may be free of contaminant proteins, which can be exploited for one-step purification of a target protein from raw cell extract. In this paper we have identified several fusion tags that can direct the elution of the target protein to the low background region of the Zn(II)-IDA elution profile. These tags allow targeting of proteins to different regions of the elution profile, facilitating purification under mild conditions.

Coordination of two high-affinity hexamer peptides to copper(II) and palladium(II) models of the peptide-metal chelation site on IMAC resins.

The coordination of peptides Ser-Pro-His-His-Gly-Gly (SPHHGG) and (His)6 (HHHHHH) to [PdII(mida)(D2O)] (mida2- = N-methyliminodiacetate) was studied by 1H NMR as model reactions for CuII(iminodiacetate)-immobilized metal affinity chromatography (IMAC) sites. This is the first direct physical description of peptide coordination for IMAC. A three-site coordination is observed which involves the first, third, and fourth residues along the peptide chain. The presence of proline in position 2 of SPHHGG achieves the best molecular mechanics and bonding angles in the coordinated peptide and enhances the interaction of the serine amino nitrogen. Histidine coordination of H1, H3, and H4 of (His)6 and H3 and H4 of SPHHGG was detected by 1H NMR contact shifts and H/D exchange of histidyl protons. The EPR spectra of SPHHGG and HHHHHH attached to the [CuII(mida)] unit were obtained for additional modeling of IMAC sites. EPR parameters of the parent [Cu(mida)(H2O)2] complex are representative: gzz = 2.31; gyy = 2.086; gxx = 2.053; A parallel = 161G; AN = 19G (three line, one N coupling). Increased rhombic distortion is detected relative to the starting aqua complex in the order of [Cu(mida)L] for distortion of HHHHHH > SPHHGG > (H2O)2. The lowering of symmetry is also seen in the decrease in the N-shf coupling, presumably to the imino nitrogen of mida2- in the order 19 G (H2O), 16 G (SPHHGG) and 11 G (HHHHHH). Visible spectra of the [Cu(mida)(SPHHGG)] and [Cu(mida)(HHHHHH)] as a function of pH indicate coordination of one histidyl donor at ca. 4.5, two in the range of pH 5-7, and two chelate ring attachments involving the terminal amino donor for SPHHGG or another histidyl donor of HHHHHH in the pH domain of 7-8 in agreement with the [PdII(mida)L] derivatives which form the two-chelate-ring attachment even at lower pH as shown by the 1H NMR methods.

Developing Carrier Complexes for "Caged NO": RuCl(3)(NO)(H(2)O)(2) Complexes of Dipyridylamine, (dpaH), N,N,N'N'-Tetrakis (2-Pyridyl) Adipamide, (tpada), and (2-Pyridylmethyl) Iminodiacetate, (pida).

Delivery agents which can carry the {Ru(NO)}(6) chromophore ("caged NO") are desired for vasodilation and for photodynamic therapy of tumors. Toward these goals, complexes derived from [RuCl(3)(NO)(H(2)O)(2)]= (1) have been prepared using dipyridylamine (dpaH) as mono and bis adducts, [Ru(NO)Cl(3)(dpaH)] = (2) and [Ru(NO)Cl(dpaH)(2)]Cl(2) = (3). The dpaH ligands coordinate cis to the Ru(NO) axis.The mono derivative is a model for a potential DNA groove-spanning binuclear complex {[RuNO)Cl(3)](2)(tpada)} = (4) which has two DNA-coordinating Ru(II) centers, photo-labile {Ru(NO)}(6) sites, and a groove-spanning tether moiety.The binuclear assembly is prepared from the tethered dipyridylamine ligand N,N,N',N'-tetrakis(2-pyridylmethyl)adipamide (tpada) which has recently been shown to provide a binuclear carrier complex suited to transporting Ru(II) and Pd(II) agents. A related complex, [Ru(NO)Cl(pida)] = (5) with the {Ru(NO)}(6) moiety bound to (2-pyridylmethyl) iminodiacetate (pida(2-)) is also characterized as a potential "caged NO" carrier. Structural information concerning the placement of the pyridyl donor groups relative to the {Ru(NO)}(6) unit has been obtained from (1)H and (13)C NMR and infrared methods, noting that a pyridyl donor trans to NO+ causes "trans strengthening" of this ligand for [Ru(NO)Cl(pida)], whereas placement of pyridyl groups cis to NO+ causes a weakening of the N-O bond and a lower NO stretching frequency in the dpa-based complexes.

Prior exercise suppresses the plasma tumor necrosis factor response to bacterial lipopolysaccharide.

This study was initiated to determine the effect of physical exercise on the in vivo tumor necrosis factor-alpha (TNF) response to Escherichia coli lipopolysaccharide (LPS). Rats familiarized with treadmill running and surgically implanted with vascular catheters were either not exercised or exercised to near exhaustion (mean run time of 102 +/- 13 min) before intravenous LPS challenge (1 mg/kg; lethality of dose is 10-20% in 24 h). Compared with time-matched nonexercised control rats, exercised rats had increased heart rates, plasma lactate, and plasma corticosterone and decreased plasma glucose at the conclusion of exercise. In response to LPS, both groups became hypotensive, exhibited transient hyperglycemia, and sustained hyperlactacidemia. By 30 min post-LPS, plasma corticosterone levels were similar in the two groups. Nonexercised rats exhibited a normal plasma TNF response to LPS with the peak value (10,400 +/- 2,000 U/ml) occurring 90 min after LPS challenge. In contrast, the TNF response in rats exercised before LPS administration was blunted to 17% of the nonexercised group, with the peak occurring at an earlier time after LPS. Addition of recombinant murine TNF to postexercise plasma was fully expressed. The TNF response remained attenuated when LPS was administered up to 6 h after completion of exercise, but it returned to normal in rats allowed to recover for 24 h. The results demonstrate that exercise, perhaps as a stress modality, markedly suppresses the systemic TNF response that is normally observed in response to LPS challenge.

Exercise training improves cardiac performance in diabetic rats.

Diabetes mellitus is often associated with a cardiomyopathy characterized by alterations in cardiac metabolism and declines in cardiac performance. We sought to determine whether exercise training would attenuate the depressed cardiac performance seen in diabetic animals. Female rats were divided into four groups: sedentary control, trained control, sedentary diabetics, and trained diabetics. After 1 week of training, we induced diabetes by intravenous injection of streptozotocin (65 mg/kg). We trained animals on a treadmill using a progressive protocol that plateaued at 27 m/min for 1 hr/day, 5 days/week for a total of 8 weeks. We measured cardiac output at a variety of left atrial filling pressures with an isolated working heart apparatus; glucose was the sole metabolic substrate for the heart. Training increased succinate dehydrogenase activity in the soleus muscle of exercised rats, but did not change heart and body weights or plasma glucose and thyroid hormone levels. The diabetic groups exhibited depressed cardiac outputs at high workloads compared to nondiabetics. Training increased the cardiac output of both sedentary and diabetic animals at high, but not low, preloads. We suggest that exercise can attenuate the severity of diabetic cardiomyopathy.

Lactobionic and gluconic acid complexes of FeII and FeIII; control of oxidation pathways by an organ transplantation preservant.

Lactobionic acid, [4-beta-(galactosido)-D-gluconic acid] = LBA, is the major component of the Wisconsin organ transplantation preservant fluid and may suppress oxygen radical-induced tissue damage upon reperfusion by the control of FeII autoxidation. FeII and FeIII complexes of LBA and the related gluconic acid (GLC) have been studied herein by titrimetric, infrared, and electrochemical methods (CV; DPP). FeII(GLC) forms quickly at pH 7, but FeII(LBA) reacts in two steps, the second requiring 4 hr. The initial complex lacks coordination of the LBA carboxylate (C-1) and is bound by the "2,3,5" hydroxyl groups. The slow rearrangement forms a "1,2,3,6" chelate which FeII(LBA) shares in common with the donor set of the FeIII(LBA) complex. Titration data shows the removal of three protons from LBA through pH 5 and an additional proton from pH 6 to 9 which is indicative of the [FeIII(LBA)(OH)(H2O)]- formulation with LBA donating at the "1,2,3,6" positions. The more stable, second form of FeII(LBA) has been investigated in its oxidation mechanisms with H2O2 and O2 using selected trapping agents for HO. and ferryl intermediates. Eighty-six percent of the oxidation events of FeII(LBA)/H2O2 occurs in steps involving formation and reduction of freely diffusible HO.. These pathways are altered by the known HO. traps t-butanol, dmso, ethanol, and methanol in the manner predictable for beta-oxidizing radicals (from t-butanol or dmso) and alpha-reducing radicals (from ethanol and methanol). Fourteen percent of the FeII(LBA)/H2O2 reaction occurs via FeIVO intermediates not trapped by t-butanol or dmso, but intercepted by primary and secondary alcohols. The HO. generating pathways are responsible for a competitive LBA ligand oxidation at the C-2 position via HO., formed from FeII(LBA) and H2O2 within the original reaction cage. Competitive ligand oxidation at C-2 is absent for the FeII(LBA)/O2 autoxidation, indicative of a different redox mechanism. The FeII(LBA)/O2 reaction rate is first-order in each component and is insensitive to the presence of t-butanol as an HO. trap. These observations support a ferryl intermediate in the autoxidation pathway and the absence of HO. or free H2O2 during autoxidation. Although chelation of FeII by hard ligand donors such as edta4-, Cl-, or HPO4(2-) accelerate the rate of autoxidation of FeII, chelation of carboxylate, alkoxy, and hydroxyl donors of LBA does not accelerate autoxidation. The implications of these findings, and the absence of an inner-sphere coordination role of the 4-beta-(galactosido) functionality toward the action of LBA in organ preservant fluids, are discussed.

Differential regulation of glucose transporter gene expression in adipose tissue or septic rats.

To understand the molecular mechanisms responsible for the sepsis-induced enhanced glucose uptake, we have examined the levels of GLUT4 and GLUT1 mRNA and protein in the adipose tissue of septic animals. Rats were challenged with a nonlethal septic insult where euglycemia was maintained and hexose uptake in adipose tissue was markedly elevated. Northern blot analysis of total RNA isolated from epididymal fat pads indicated differential regulation of the mRNA content for the two transporters: GLUT1 mRNA was increased 2.6 to 4.6-fold, while GLUT4 mRNA was decreased by 2.5 to 2.9-fold. Despite the difference in mRNA levels, both GLUT1 and GLUT4 protein were down regulated in plasma membranes (40% and 25%, respectively) and microsomal membranes (42% and 25%, respectively) of the septic animals. The increased glucose uptake cannot be explained by the membrane content of GLUT1 and GLUT4 protein. Thus, during hypermetabolic sepsis, increased glucose utilization by adipose tissue is dependent on alternative processes.

Exercise training attenuates the myocardial dysfunction induced by endotoxin.

The purpose of this study was to determine whether exercise training protected against endotoxin-induced myocardial dysfunction. After a 12-wk treadmill training period, carotid catheters were implanted 24 h before saline or endotoxin administration into four groups of animals: trained saline-injected (TS), trained endotoxin-injected (TE), sedentary saline-injected (SS), and sedentary endotoxin-injected (SE). Heart rate and mean arterial pressure were monitored 4 h after in vivo endotoxin or saline injection. Mean arterial pressure decreased an average of 32 +/- 3 mmHg 1 h after endotoxin administration but was normal (109 +/- 6 mmHg) 2 h later. Plasma catecholamines, in vitro myocardial performance, and isolated myocyte adenosine 3',5'-cyclic monophosphate (cAMP) production in response to isoproterenol were assessed 4 h after endotoxin injection. Plasma catecholamine levels were 5- to 15-fold higher in SE compared with the other groups. These data suggest that myocardial protection may be related to the lowered catecholamine levels elicited in TE compared with SE in response to endotoxin administration. The product of cardiac output and peak systolic pressure, an index of cardiac work, was 24-32% greater in TS compared with SS. Cardiac work was decreased 32% in TE compared with a 45% decrease in SE. cAMP was reduced in myocytes from SE in response to isoproterenol (-28%) and to forskolin (-44%) but not in myocytes from TE, compared with TS and SS. The difference in cAMP accumulation suggests that training maintains the integrity of the beta-adrenergic receptor adenylate cyclase system, which can be depressed by in vivo endotoxin administration.

Lactobionic acid as an iron chelator: a rationale for its effectiveness as an organ preservant.

Lactobionic acid, a major constituent of a solution used to preserve organs prior to transplantation, can chelate ferric iron. This is evident by its ability to solubilize iron as well as changes that occur in the UV-VIS spectra of iron in its presence. Relative to iron (III) chelated to EDTA, the lactobionic acid-iron (III) complex is less able to participate in the Fenton reaction as measured by formaldehyde generation from DMSO and bleaching of p-N,N-dimethylnitrosoaniline. Similar effects are seen with citrate and ATP, two substances which also appear to be able to ameliorate ischemia/reperfusion injury. These findings present a rationale for the effectiveness of lactobionic acid as an organ preservant.

The effect of dichloroacetate on the isolated no flow arrested rat heart.

Ischemic dysfunction, including contracture, has been attributed to lack of ATP, although previous work has not been consistent with this concept. We describe here a model of no flow ischemic arrest, characterized by depressed levels of mechanical function upon reperfusion and high energy phosphate stores within normal limits. The decreased mechanical function bears an inverse relationship to myocardial lactate levels after twenty-minutes of reperfusion in the absence or presence of dichloroacetic acid (DCA). Post-ischemic non-DCA treated hearts attained peak work of only 25% of that of controls, while those treated with DCA following ischemia performed almost as well as controls. ATP and CP levels remained high in both DCA treated and non-DCA treated hearts. Lactate levels were high in hearts immediately following ischemia, but were reduced to control levels in post-ischemic hearts perfused with DCA within twenty minutes, whereas those not treated with DCA had lactate levels two to three times that of controls within the same time period. Pyruvate dehydrogenase (PDH) activity was reduced in non-DCA treated post ischemic hearts after twenty minutes reperfusion but was elevated above controls in hearts reperfused with DCA. The data indicates that DCA increases mechanical performance of the isolated post-ischemic rat heart and the proposed mechanism for this increase is the oxidative removal of lactate resulting from an increase in PDH activity.

Cyclic AMP regulation of fuel metabolism during exercise: regulation of adipose tissue lipolysis during exercise.

Adipocytes from trained rats release more free fatty acids in response to hormonal challenge compared to fat cells from sedentary rats. Lipolysis results from increased triglyceride hydrolysis that is catalyzed by a hormone-sensitive lipase, which, in turn, is activated by a phosphorylation mechanism involving cyclic AMP-dependent protein kinase. Cyclic AMP levels within the fat cell are regulated by beta-adrenergic receptor/adenylate cyclase interactions and cyclic AMP phosphodiesterase activity. This review focuses on cyclic AMP regulation of lipolysis in adipocytes from trained and sedentary animals. Although lipolysis is elevated in fat cells from trained rats, no differences are found in beta-adrenergic receptor number or affinity, adenylate cyclase activity, protein kinase activity, or partially purified hormone-sensitive lipase activity when compared to sedentary rats. The major lipolytic alteration induced by exercise training appears to occur at a site distal to hormonal regulation of the beta-adrenergic receptor.

Inotropic interactions of dichloroacetate with amrinone and ouabain in isolated hearts from endotoxin-shocked rats.

The inotropic effects of ouabain and amrinone singly and in combination with dichloroacetate (DCA) were assessed using isolated working perfused hearts from endotoxin-shocked (LD50/6 h) rats, with glucose and free fatty acids as substrates. Amrinone (2.7 X 10(-4) M) and ouabain (10(-5) M) alone improved myocardial mechanical performance from 25 to 75%, depending on the preload. Amrinone was more effective than ouabain at all left atrial filling pressures tested. DCA enhanced the inotropic effect of both compounds, improving myocardial work greater than 65% in combination with ouabain (at low filling pressures) to 125% with amrinone (at high filling pressures). Glucose oxidation rose two- to threefold when DCA was present with either drug. Ouabain was without effect on pyruvate dehydrogenase (PDH) activity; however, when present with DCA, PDH activity increased fourfold. Amrinone alone augmented PDH activity 2.5 times as compared with controls, and 4.5 times when combined with DCA. All three compounds individually elevated myocardial ATP levels, but in contrast to the inotropic effects, when used in combination caused no further increase in ATP. Myocardial cyclic AMP (cyclic AMP) levels were augmented five times control values in the presence of amrinone. The simultaneous presence of DCA and amrinone did not further augment the myocardial concentration of cAMP. DCA alone enhanced the myocardial oxidation of glucose in isolated myocardial cells from endotoxin-shocked animals. In contrast, ouabain and amrinone did not affect cellular glucose oxidation. These data indicate that the provision of DCA can enhance the inotropic effect of amrinone and ouabain on the isolated working heart.(ABSTRACT TRUNCATED AT 250 WORDS)

T3 treatment does not prevent myocardial dysfunction in chronically diabetic rats.

The isolated working heart preparation was used to investigate the effect of continuous triiodothyronine (T3) administration on cardiac function and metabolism of rats rendered diabetic for a period of 4 wk with streptozocin (STZ). T3 controlled-release pellets were implanted 1 wk after STZ (70 mg/kg) injection. Rats injected with citrate buffer without STZ received T3 pellets 1 and/or 2 wk later. A comparable number of rats received placebo pellets. Untreated diabetic rats exhibited a decrease in spontaneous heart rate and myocardial cytochrome c concentrations concurrent with depressed plasma T3 values compared with untreated controls. T3 treatment did not improve in vitro cardiac performance (assessed as cardiac output times peak systolic pressure per gram dry heart weight) in hearts from diabetic rats perfused with glucose alone. Addition of octanoate reversed this depression and improved cardiac function to a greater extent in treated than in untreated diabetic animals. However, these differences between treated and untreated diabetic animals disappeared when heart rate was controlled by cardiac pacing. Furthermore, T3 treatment of controls and diabetics did not alter the oxidation of octanoate or the cardiac responsiveness to isoproterenol. These results suggest that experimental diabetic cardiomyopathy is partly attributable to a substrate deficiency and is not due entirely to hypothyroidism.

Lipolysis and beta-adrenergic receptor binding on adipocytes of spontaneously hypertensive rats.

Adipocytes from spontaneously hypertensive rats demonstrated a blunted lipolytic response to isoproterenol and dibutyryl cyclic AMP. (-)-[3H]Dihydroalprenolol binding was examined in adipocytes from normotensive and spontaneously hypertensive rats. Increasing concentrations of isoproterenol decreased total (-)-[3H]dihydroalprenolol binding to intact cells from normotensive rats, and the efficacy of competition was decreased in adipocytes from spontaneously hypertensive rats. Scatchard analysis indicated that the number of (-)-[3H]dihydroalprenolol binding sites and the affinity of dihydroalprenolol binding were comparable between normotensive and spontaneously hypertensive rats. Isoproterenol- and Gpp(NH)p-stimulated adenylate cyclase activity was consistently depressed in adipocyte membranes from spontaneously hypertensive rats as compared to normotensive rats. No difference in fluoride-stimulated adenylate cyclase activity was observed. The blunted lipolytic and cyclic AMP response to isoproterenol in these cells suggest a postreceptor lesion of the lipolytic pathway (possibly the guanine nucleotide regulatory protein) in adipocytes from spontaneously hypertensive rats. The blunted lipolytic response to dibutyryl cyclic AMP suggests defective regulation of lipolytic enzymes at the protein kinase-hormone-sensitive lipase level.

Myocardial adrenergic responsiveness after lethal and nonlethal doses of endotoxin.

A dose-dependent impairment of intrinsic myocardial performance has been observed following in vivo administration of endotoxin. The present study reports a dose-dependent increase in plasma catecholamines following endotoxin (ET) that may impair beta-adrenergic responsiveness. Hearts were removed from pentobarbital-anesthetized rats 4 h after a bolus injection of saline or ET (1,000, 100, or 10 micrograms/100 g body wt) and were studied as isolated cell preparations following collagenase digestion. Responsiveness of isoproterenol-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in myocytes prepared from hearts of animals injected with 10 and 100 micrograms ET was decreased when compared with control rats and was significantly blunted in myocytes prepared from animals receiving 1,000 micrograms ET. Similar sensitivities of the cAMP system existed, as judged by similar half-maximum effective concentration values. cAMP accumulation in the presence of 1 microM forskolin was depressed in myocytes from the 1,000-microgram ET animals; beta-adrenergic receptor density was decreased 25% (P less than 0.05) in myocytes from high-dose ET animals when compared with control animals. This was accompanied by a nonsignificant reduction in the affinity of binding sites for (+/-) [3H]CGP 12177. The blunted myocyte hormonal responsiveness following ET challenge appears to be related to the decreased activity of the adenylate cyclase that may be attributed to alterations in both receptor density and in the adenylate cyclase itself.

Enhanced lipolysis is not evident in adipocytes from exercise-trained SHR.

Adipocytes from spontaneously hypertensive rats (SHR) are not as responsive to isoproterenol or dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) stimulation compared with Sprague-Dawley or Wistar-Kyoto rats. Lipolytic activity in adipocytes from trained normotensive rats was enhanced in response to 1 microM isoproterenol and 0.5 mM dibutyryl cAMP but not in adipocytes from trained SHR. Decreases in isoproterenol-stimulated (1 microM) cAMP accumulation were evident in adipocytes from trained normotensive rats but not in adipocytes from trained SHR. Basal and agonist-induced lipolysis in fat cells isolated from both normotensive rats and SHR immediately following a 60-min run was increased in both sedentary and trained rats. Adenylate cyclase activity in fat cell membranes was blunted in sedentary and trained SHR both in the absence and presence of 100 microM 5'-guanylyl imidophosphate. No apparent differences existed in antagonist affinity of binding sites for the antagonist dihydroalprenolol in normal rats or SHR. Evidence for a change in affinity of agonist isoproterenol might be indicated based on the enhanced potency of isoproterenol to stimulate lipolysis in trained normal rats. beta-Adrenergic receptor density and antagonist affinity were not different in normotensive rats and SHR in response to training. However, displacement of [3H]dihydroalprenolol in adipocytes from SHR required greater concentrations of isoproterenol compared with adipocytes from normotensive rats, further suggestive of increased agonist affinity of binding sites in normal rats. These data suggest a postreceptor lesion of the lipolytic pathway in adipocytes from spontaneously hypertensive rats, possibly at the guanine nucleotide regulatory protein level.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of cyclic AMP phosphodiesterase in adipose tissue from trained rats.

Fat cells were isolated from sedentary and exercise trained female Sprague-Dawley rats and cyclic AMP phosphodiesterase (cyclic AMP-PDE) activities were determined from crude homogenates of the fat cells in the whole homogenate, P5, P48, and S48 fractions. Exercise training resulted in a significant increase in the mean specific activity of cyclic AMP-PDE (pmol X min-1 X mg-1) from the whole homogenate and S48 fraction at cyclic AMP concentrations of 4, 8, and 16 microM and in the P48 fraction at 8 and 16 microM cyclic AMP. Cyclic AMP-PDE kinetic plots according to Lineweaver-Burk for the calculation of Michaelis constants (Km) and maximum enzyme velocities (Vmax) were nonlinear, indicating both a low and high enzyme form. The Michaelis constants were significantly lower in trained rats than those of its control for the high Km form of cyclic AMP-PDE in the whole and soluble fractions and for the low Km form of the P5 particulate fraction. The Vmax of the high Km form of the P48 particulate fraction from trained animals was also significantly higher than that found in its control. Phosphodiesterase inhibition by methylxanthines in the various fractions was similar in both trained and sedentary animals. These changes in specific activity, Michaelis constants, and Vmax of cyclic AMP-PDE from crude homogenates of isolated fat cells from exercise trained animals may account for the decreased intracellular levels of cyclic AMP following catecholamine stimulation of isolated fat cells from trained rats.

Muscle lipoprotein lipase activity in voluntarily exercising rats.

Voluntary exercise of rats in freely rotating work wheels has been extensively used, but muscle adaptations that result from such exercise training are poorly documented. The purpose of this study was to determine whether the exercise performed by voluntarily active rats would increase succinate dehydrogenase or lipoprotein lipase activities in the soleus muscle (SM) or the red portion of the vastus lateralis muscle (RV). In SM the activities of these two enzymes were not increased after 7 or 16 wk of voluntary exercise. Succinate dehydrogenase activity in RV was moderately increased after 7 and 16 wk of voluntary activity (P less than 0.05). Substantial increases occurred in RV lipoprotein lipase activity (P less than 0.01). The increase in RV lipoprotein lipase activity was positively related to distance run by the rats. The results indicate that only small muscle-dependent increases in mitochondrial enzymes occur in rats allowed to exercise voluntarily in rodent work wheels. Voluntary exercise training induced a selective increase in lipoprotein lipase activity in a muscle containing a high percentage of fast-twitch red fibers, a response absent in a muscle containing a predominance of slow-twitch red fibers. It is unlikely that this differential response can be explained by exercise-induced changes in plasma hormone concentrations involved in the regulation of lipoprotein lipase.