Rat heart is a site of leptin production and action

Leptin, the 16-kDa peptide hormone product of the ob gene, is produced primarily by adipocytes and was initially thought to exert its effects exclusively through actions on the hypothalamus via distinct leptin receptors termed OB-R. However, recent data show that leptin is produced elsewhere and that receptors are present in many other tissues. Using real-time PCR, we determined whether leptin and its receptors are present in the rat heart and demonstrated regional distribution patterns and gender differences as well as the effect of ischemia and reperfusion. Gene expression of leptin and its receptors (OB-Ra, OB-Rb, and OB-Re) was identified in myocytes and whole heart homogenates from all regions of the heart of male and female rats, with the highest abundance in left and right atria of male and female rats, respectively. No differences in regional distribution of OB-R were evident in male rat hearts. In female rats, expression was highest in right atria for all three isoforms and was significantly greater than in male rats. Ischemia and reperfusion significantly downregulated leptin and OB-R expression, although this was more pronounced in male rat hearts. Leptin release in the coronary effluent was also detected using ELISA, although this was generally unaffected by global ischemia and reperfusion. Our results demonstrate for the first time the presence of the leptin system, including the peptide and its receptors, in all regions of the rat heart. In view of emerging evidence for cardiac effects of leptin, it is proposed that the heart is a target for leptin action and that the peptide modulates function through a paracrine- or autocrine-dependent manner.     

RGS2 is upregulated by and attenuates the hypertrophic effect of alpha1-adrenergic activation in cultured ventricular myocytes

Regulator of G protein signaling (RGS) proteins counter the effects of G protein-coupled receptors (GPCRs) by limiting the abilities of G proteins to propagate signals, although little is known concerning their role in cardiac pathophysiology. We investigated the potential role of RGS proteins on alpha1-adrenergic receptor signals associated with hypertrophy in primary cultures of neonatal rat cardiomyocytes. Levels of mRNA encoding RGS proteins 1-5 were examined, and the alpha1-adrenergic agonist phenylephrine (PE) significantly increased RGS2 gene expression but had little or no effect on the others. The greatest changes in RGS2 mRNA occurred within the first hour of agonist addition. We next investigated the effects of RGS2 overexpression produced by infecting cells with an adenovirus encoding RGS2-cDNA on cardiomyocyte responses to PE. As expected, PE increased cardiomyocyte size and also significantly upregulated alpha-skeletal actin and ANP expression, the markers of hypertrophy, as well as the Na-H exchanger 1 isoform. These effects were blocked in cells infected with the adenovirus expressing RGS2. We also examined hypertrophy-associated MAP kinase pathways, and RGS2 overexpression completely prevented the activation of ERK by PE. In contrast, the activation of both JNK and p38 unexpectedly were increased by RGS2, although the ability of PE to further activate the p38 pathway was reduced. These results indicate that RGS2 is an important negative-regulatory factor in cardiac hypertrophy produced by alpha1-adrenergic receptor stimulation through complex mechanisms involving the modulation of mitogen-activated protein kinase signaling pathways.     

RGS2 inhibits β-adrenergic receptor-induced cardiomyocyte hypertrophy

The chronic stimulation of certain G protein-coupled receptors promotes cardiomyocyte hypertrophy and thus plays a pivotal role in the development of human heart failure. The beta-adrenergic receptors (β-AR) are unique among these in that they signal via Gs, whereas others, such as the alpha1-adrenergic (α1-AR) and endothelin-1 (ET-1) receptors, predominantly act through Gq. In this study, we investigated the potential role of regulator of G protein signalling 2 (RGS2) in modulating the hypertrophic effects of the β-AR agonist isoproterenol (ISO) in rat neonatal ventricular cardiomyocytes. We found that ISO-induced hypertrophy in rat neonatal ventricular myocytes was accompanied by the selective upregulation of RGS2 mRNA, with little or no change in RGS1, RGS3, RGS4 or RGS5. The adenylyl cyclase activator forskolin had a similar effect suggesting that it was mediated through cAMP production. To study the role of RGS2 upregulation in β-AR-dependent hypertrophy, cardiomyocytes were infected with adenovirus encoding RGS2 and assayed for cell growth, markers of hypertrophy, and β-AR signalling. ISO-induced increases in cell surface area were virtually eliminated by the overexpression of RGS2, as were increases in α-skeletal actin and atrial natriuretic peptide. RGS2 overexpression also significantly attenuated ISO-induced extracellular signal-regulated kinases 1 and 2 (ERK1/2) and Akt activation, which may account for, or contribute to, its observed antihypertrophic effects. In contrast, RGS2 overexpression significantly activated JNK MAP kinase, while decreasing the potency but not the maximal effect of ISO on cAMP accumulation. In conclusion, the present results suggest that RGS2 negatively regulates hypertrophy induced by β-AR activation and thus may play a protective role in cardiac hypertrophy.     

Up-regulation of endogenous RGS2 mediates cross-desensitization between Gs and Gq signaling in osteoblasts

Regulator of G protein signaling (RGS) proteins limit G protein signals. In this study, we investigated the role of RGS2 in the control of G protein signaling cascades in osteoblasts, the cells responsible for bone formation. Expression of RGS2 was up-regulated in primary cultures of mouse calvarial osteoblasts by parathyroid hormone-related peptide (PTHrP)-(1-34), which stimulates G(s) signaling. RGS2 was also up-regulated by extracellular ATP, which selectively activates G(q), as well as by forskolin and phorbol myristate acetate, which activate targets downstream of G(s) and G(q), respectively. To assess the role of endogenous RGS2, we characterized G(s) and G(q) signaling in osteoblasts derived from wild type and rgs2(-/-) mice. Under control conditions, nucleotide-stimulated calcium release, endothelin-stimulated accumulation of inositol phosphates, and PTHrP-stimulated cAMP accumulation were equivalent in osteoblasts isolated from wild type and rgs2(-/-) mice. Thus, basal levels of endogenous RGS2 do not appear to regulate G(s) or G(q) signaling in osteoblasts. Interestingly, forskolin treatment of wild type but not rgs2(-/-) osteoblasts suppressed both endothelin-stimulated accumulation of inositol phosphates and nucleotide-stimulated calcium release, indicating that up-regulation of RGS2 by G(s) signaling desensitizes G(q) signals. Furthermore, pretreatment with ATP suppressed PTHrP-dependent cAMP accumulation in wild type but not rgs2(-/-) osteoblasts, implying that up-regulation of RGS2 by G(q) signaling desensitizes G(s) signals. Our findings demonstrate that endogenously expressed RGS2 can limit G(s) signaling. Moreover, up-regulation of RGS2 contributes to cross-desensitization of G(s)- and G(q)-coupled signals.     

Resistance to age-related, normal body weight gain in RGS2 deficient mice

RGS2 (regulator of G protein signaling 2) is known to limit signals mediated via Gq- and Gs-coupled GPCRs (G protein coupled receptors), and it has been implicated in the differentiation of several cells types. The physiology of RGS2 knockout mice (rgs2^−/−) has been studied in some detail, however, a metabolic phenotype has not previously been reported. We observed that old (21-24 month) rgs2^−/− mice weigh much less than wild-type C57BL/6 controls, and exhibit greatly reduced fat deposits, decreased serum lipids, and low leptin levels. Lower weight was evident as early as four weeks and continued throughout life. Younger adult male rgs2^−/− mice (4-8 months) were found to show similar strain-related differences as the aged animals, as well improved glucose clearance and insulin sensitivity, and enhanced beta-adrenergic and glucagon signaling in isolated hepatocytes. In addition, rgs2^−/− pre-adipocytes had reduced levels of differentiation markers (Peroxisome proliferator-activated receptor γ (PPARγ); lipoprotein lipase (Lpl); CCAAT/enhancer binding protein α (CEBPα)) and also rgs2^−/− white adipocytes were small relative to controls, suggesting altered adipogenesis. In wild-type animals, RGS2 mRNA was decreased in brown adipose tissue after cold exposure (7 h at 4 °C) but increased in white adipose tissue in response to a high fat diet, also suggesting a role in lipid storage. No differences between strains were detected with respect to food intake, energy expenditure, GPCR-stimulated lipolysis, or adaptive thermogenesis. In conclusion this study points to RGS2 as being an important regulatory factor in controlling body weight and adipose function.     

Pre- and postsynaptic mechanisms in Hebbian activity-dependent synapse modification

We have used a three compartment tissue culture system that involved two separate populations of cholinergic neurons in the side compartments that converged on a common target population of myotubes in the center compartment. Activation of the axons from one population of neurons produced selective down-regulation of the synaptic inputs from the other neuronal population (when the two inputs innervated the same myotubes). The decrease in heterosynaptic inputs was mediated by protein kinase C (PKC). An activity-dependent action of protein kinase A (PKA) was associated with the stimulated input and this served to selectively stabilize this input. These changes associated with PKA and PKC activation were mediated by alterations in the number of acetylcholine receptors at the neuromuscular junction. These results suggest that neuromuscular electrical activity produces postsynaptic activation of both PKA and PKC, with the latter producing generalized synapse weakening and the former a selective synapse stabilization. Treatment of the neuronal cell body and axon to increase PKC activity by putting phorbal ester (PMA) in the side chamber did not affect synaptic transmission (with or without stimulation). By contrast, PKA blockade in the side compartment did produce an activity-dependent decrease in synaptic efficacy, which was due to a decrease in quantal release of neurotransmitter. Thus, when the synapse is activated, it appears that presynaptic PKA action is necessary to maintain transmitter output.     

Phosphorylation reactions in activity-dependent synapse modification at the neuromuscular junction during development

We have studied developmental activity-dependent synapse diminution in both an in vitro tissue culture chamber system and at the intact rodent neuromuscular junction (nmj). In both types of preparations, pre- and postsynaptic alterations in synapse structure and function are produced by manipulations of thrombin (Thr) and protein kinase C (PKC) activity. An opposing postsynaptic effect of PKC and protein kinase A (PKA) action on the acetycholine receptor (AChR) can be shown in vitro with PKA stabilizing and PKC destabilizing the nmj synapses. In vivo studies of normal junctional maturation show that changes in axonal inputs and postsynaptic receptor cluster morphology occur, to a substantial degree, independently of one another. Presynaptic actions of PKA are involved in the activity dependent synapse modulation that can be demonstrated in vitro. Late in the elimination process, (>12 days in vivo) the process becomes independent of PKC, implying that diverse, redundant mechanisms are involved in this important developmental process.     

Extracellular ATP in activity-dependent remodeling of the neuromuscular junction

Electrical activity during early development affects the development and maintenance of synapses (Spitzer [2006]: Nature 4447:707-712), but the intercellular signals regulating maintenance of synapses are not well identified. At the neuromuscular junction, adenosine 5-triphosphate (ATP) is coreleased with acetylcholine at activated nerve terminals to modulate synaptic function. Here we use cocultured mouse motor neurons and muscle cells in a three-compartment cell culture chamber to test whether endogenously released ATP plays a role in activity-dependent maintenance of neuromuscular synapses. The results suggest that ATP release at the synapse counters the negative effect of electrical activity, thus stabilizing activated synapses. Confirming our previous work (Li et al. [2001]: Nat Neurosci 4:871-872), we found that in doubly innervated muscles, electrical stimulation induced heterosynaptic downregulation of the nonstimulated convergent input to the muscle fiber with no or little change of the stimulated inputs. However, in preparations that were stimulated in the presence of apyrase, an enzyme that degrades extracellular ATP, synapse downregulation of stimulated inputs was substantial and significant, and end plate potentials were reduced. Apyrase treatment for 20 h in the absence of stimulation did result in moderate diminution, but this was prevented by blocking spontaneous neural activity with tetrodotoxin. The P2 receptor blocker, suramin, also induced activity-dependent synapse diminution. The decrease in synaptic efficacy produced by prolonged stimulation in the presence of apyrase persisted for greater than 20 h, consistent with a developmental time-course and distinct from the rapid neuromodulatory actions of ATP that have been demonstrated by others. We conclude that extracellular ATP promotes stabilization of the neuromuscular junction and may play a role in activity-dependent synaptic modification during development.