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Heart failure is accompanied by electrolyte disturbance including reduced calcium and sodium in the extracellular milieu but increased calcium within cells, a phenomenon called "calcium paradox". Aldosteronism is considered as part of this disorder. Aldosterone antagonism is known to reduce cardiac mortality on top of standard therapies such as antagonism of the renin-angiotensin-system. However, the effect of aldosterone on cardiac function under basal conditions and conditions more closely related to those seen in heart failure remains elusive. In order to address this question the function of isolated cardiomyocytes was determined as unloaded cell shortening. Cardiomyocytes were isolated from adult rat hearts and cultured for 24 h in the presence of aldosterone. Thereafter, cell shortening was determined in cells that were electrically paced (0.5-2.0 Hz). The effect of aldosterone on cell shortening was investigated under basal and maximal inotropic stimulation, preincubation with angiotensin II and myocytes from spontaneously hypertensive rats. The composition of the culture medium was modified according to the extracellular milieu found in patients with end-stage heart failure. Aldosterone increased cell shortening in a frequency-dependent way under basal conditions and conditions of low calcium. It potentiated the effect of beta-adrenoceptor stimulation, increased the formation of oxygen radicals, and increased diastolic and systolic calcium. In conclusion, chronic exposure to aldosterone improves the function of cardiomyocytes under basal conditions and electrolyte disturbances that mimic the situation found in heart failure patients.

Aims: In patients with congestive heart failure, plasma parathyroid hormone (PTH) levels are positively associated with cardiac function. PTH, used to mobilize stem cells from the bone marrow after myocardial infarction, causes an increased left ventricular ejection fraction. The aim of this study was to investigate whether low but plasma-relevant concentrations of PTH directly influence the contractile properties of cardiomyocytes., Methods and Results: Isolated adult rat ventricular cardiomyocytes were exposed to PTH(1-34) or full-length PTH at picomolar concentrations for 24 h. Cell shortening was measured at 2 Hz as a cellular correlate of inotropic responsiveness. Intracellular calcium was measured in Fura-AM-loaded cells. PTH(1-3) (20-200 pM) and full-length PTH (200 pM) increased cell shortening within 24 h. PTH had no effect on cell size, but resting and peak systolic calcium concentrations were elevated. The beneficial effect of PTH was mediated via its cAMP/protein kinase A-activating domain and attenuated by addition of a protein kinase A inhibitor. In contrast, PTH peptides representing a protein kinase C-activating domain but not a cAMP/protein kinase A-activating domain or peptides that represent none of these domains had no effect on cell shortening. The effect of PTH on cell shortening was strong at low concentrations of extracellular calcium but declined at higher calcium concentrations. PTH downregulated the expression of the calcium sensing receptor, a receptor known to antagonize the action of PTH on calcium transport. Furthermore, PTH antagonized the angiotensin II-induced loss of cell function., Conclusion: Low concentrations of PTH improve cell shortening by increasing calcium load at rest. By this mechanism cardiomyocytes compensate reduced extracellular calcium levels as they occur in patients with heart failure.

The cloning of the so-called 'parathyroid hormone-related protein' (PTHrP) in 1987 was the result of a long quest for the factor which, by mimicking the actions of PTH in bone and kidney, is responsible for the hypercalcemic paraneoplastic syndrome, humoral calcemia of malignancy. PTHrP is distinct from PTH in a number of ways. First, PTHrP is the product of a separate gene. Second, with the exception of a short N-terminal region, the structure of PTHrP is not closely related to that of PTH. Third, in contrast to PTH, PTHrP is a paracrine factor expressed throughout the body. Finally, most of the functions of PTHrP have nothing in common with those of PTH. PTHrP is a poly-hormone which comprises a family of distinct peptide hormones arising from post-translational endoproteolytic cleavage of the initial PTHrP translation products. Mature N-terminal, mid-region and C-terminal secretory forms of PTHrP are thus generated, each of them having their own physiologic functions and probably their own receptors. The type 1 PTHrP receptor, binding both PTH(1-34) and PTHrP(1-36), is the only cloned receptor so far. PTHrP is a PTH-like calciotropic hormone, a myorelaxant, a growth factor and a developmental regulatory molecule. The present review reports recent aspects of PTHrP pharmacology and physiology, including: (a) the identification of new peptides and receptors of the PTH/PTHrP system; (b) the recently discovered nuclear functions of PTHrP and the role of PTHrP as an intracrine regulator of cell growth and cell death; (c) the physiological and developmental actions of PTHrP in the cardiovascular and the renal glomerulo-vascular systems; (d) the role of PTHrP as a regulator of pancreatic beta cell growth and functions, and, (e) the interactions of PTHrP and calcium-sensing receptors for the control of the growth of placental trophoblasts. These new advances have contributed to a better understanding of the pathophysiological role of PTHrP, and will help to identify its therapeutic potential in a number of diseases.

Cardiovascular cells (cardiomyocytes and smooth muscle cells) are target cells for parathyroid hormone (PTH) and the structurally related peptide parathyroid hormone-related peptide (PTH-rP). PTH activates protein kinase C (PKC) of cardiomyocytes via a PKC activating domain previously identified on chondrocytes. Activation of PKC leads to hypertrophic growth and re-expression of fetal type proteins in cardiomyocytes. This hypertrophic effect of PTH might contribute to left ventricular hypertrophy in hemodialysis patients with secondary hyperparathyroidism. PTH-rP is expressed in cardiovascular cells (endothelial cells and smooth muscle cells). It does not mimic the above described actions of PTH but exerts effects of its own on cardiomyocytes. These effects involve activation of protein kinase A, via a N-terminal domain distinct from that identified on PTH, and activation of PKC, via a C-terminally located domain distinct from that found on PTH. On smooth muscle cells PTH and PTH-rP reduce the influence of extracellular calcium, through cAMP-dependent mechanisms. These inhibitory effects on voltage-dependent L-type calcium channels of smooth muscle cells cause vasorelaxation. Present studies concerning cardiovascular actions of either PTH and PTH-rP suggest that increased plasma levels of PTH and PTH-rP influence cardiomyocyte and smooth muscle cell physiology. It can be assumed that PTH-rP acts as a paracrine or autocrine modulator in heart and vessels.

Ventricular cardiomyocytes have been identified as target cells for parathyroid hormone (PTH). A structurally related peptide hormone, parathyroid hormone-related peptide (PTH-rP), is expressed in the heart. In the present study, it was investigated whether PTH-rP can mimic or modify effects of PTH on cardiomyocytes. The investigated effect was induction of creatine kinase (CK) activity, which is associated with cardiac hypertrophy. PTH and PTH-rP have a similar secondary structure within the active domain 28 34, with exception of amino acid 29. At this position the hydrophilic glutamine in the PTH molecule corresponds to hydrophobic alanine in the PTH-rP molecule. Synthetic PTH or PTH-rP peptides covering domain 28 34 and recombinant full-length PTH(1 84) were used. PTH(28 48) (100 nm) induced CK activity within 24 h (123 +/- 3%; means +/- S.D., n = 4). PTH-rP(7-34) (1 nm to 1 microm) failed to induce CK activity in cardiomyocytes. Given simultaneously, PTH-rP (1 mum) reduced the stimulation of CK activity by PTH(1-84), PTH(1-34), and PTH(28-48) by 94 +/- 9, 79 +/- 8, and 69 +/- 14%, respectively (means +/- S.D., n = 4). In contrast, PTH-rP(7-34) was sufficient to stimulate proliferation of chicken chondrocytes. Thus, PTH-rP exerts different effects on cardiomyocytes and classical target cells for PTH. A synthetic hybrid peptide was synthesized, [Ala29]PTH(28-48), in which alanine replaced glutamine at position 29, as in the PTH-rP molecule. In contrast to PTH(28-48), this mutated peptide [Ala29]PTH(28-48) had no intrinsic activity but antagonized the effect of PTH(1-84) and PTH(28-48) on cardiomyocytes. The results demonstrate that on cardiomyocytes the effect of PTH can be antagonized by PTH-rP. This antagonism seems due to a hydrophobic replacement at position 29.

Adult ventricular cardiomyocytes have been identified as target cells for parathyroid hormone (PTH) but little is known about its signal transduction in these cells. In the present study the influence of PTH on cyclic AMP accumulation and the activity of protein kinase C (PKC) in cardiomyocytes was evaluated. A mid-regional synthetic fragment of PTH, PTH-(28-48), which exerts a hypertrophic effect on cardiomyocytes, increased the activity of membrane-associated PKC in a dose-dependent manner (1-100 nM). Activated membranous PKC was dependent on Ca2+ and sensitive to an inhibitor of Ca(2+)-dependent isoforms of PKC. When adenylate cyclase was stimulated by the addition of isoprenaline, a beta-adrenoceptor agonist, PTH-(28-48) antagonized cyclic AMP accumulation. This antagonistic effect of PTH-(28-48) could be mimicked by activation of PKC with a phorbol ester and inhibited by isobutylmethylxanthine, a phosphodiesterase inhibitor. An N-terminal synthetic fragment, PTH-(1-34), which includes an adenylate cyclase-activating domain, did not stimulate the accumulation of cyclic AMP in cardiomyocytes. The results demonstrate that in adult cardiomyocytes PTH (1) is able to stimulate PKC, (2) is not able to cause accumulation of cyclic AMP and (3) functionally antagonizes the effect of beta-adrenoceptor stimulation to increase cellular cyclic AMP concentrations via PKC-dependent phosphodiesterase activity.

Trophic effects of isoproterenol (Iso), norepinephrine (NE), phenylephrine (PE), and biologically active fragments of parathyroid hormone (PTH), PTH-(1-34) and PTH-(28-48), were investigated in mechanically quiescent, isolated ventricular cardiomyocytes from adult rat. In 24-h incubations in modified serum-free medium 199 incorporation of [14C]phenylalanine, changes in total protein and specific activities of cytosolic enzymes, creatine kinase (CK) and lactate dehydrogenase (LDH) were monitored. NE and PE (10 microM), but not Iso, distinctly increased phenylalanine incorporation, total cell protein, and specific activity of CK but not LDH. Induction of CK, but not LDH, was also produced by phorbol 12-myristate 13-acetate (10 nM) but not dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP, 1 mM). It was abolished by copresence of cycloheximide (35 microM) or actinomycin D (5 microM). CK-BB was the only induced isoform of CK, as shown for PE incubations. PTH-(1-34) and PTH-(28-48) (30-300 nM) had effects comparable to NE and PE. They increased phenylalanine incorporation and total protein content and induced CK but not LDH. In summary, distinct trophic effects on adult cardiomyocytes were found with alpha 1-adrenergic agonists, fragments of PTH containing the midregional amino acids 28-34, and direct activation of protein kinase C but neither beta-adrenergic agonists nor DBcAMP.

We have found, in previous studies in vitro using skeletal derived cell cultures, that mid-region fragments of human parathyroid hormone (hPTH) stimulate [3H]thymidine incorporation into DNA and increase the specific activity of the brain-type isoenzyme of creatine kinase (CK). These changes occurred without an increase in cyclic AMP formation which is linked to bone resorption. In this study, we found that the mid-region fragment hPTH-(28-48) stimulated CK activity in diaphysis, epiphysis and kidney in a time- and dose-dependent manner, parallel to the effects of the whole molecule bovine (b)PTH-(1-84) and the fully active fragment hPTH-(1-34). The increase caused by hPTH-(28-48) at a dose of 1.25 micrograms/rat was not less than the 2-fold increase caused by a roughly equimolar concentration bPTH-(1-84). A significant increase was reached at 1 h after intraperitoneal injection in all cases. All three sequences of PTH caused an increase in [3H]thymidine incorporation into DNA in diaphysis and epiphysis, but not in kidney, 24 h after injection. A fragment further towards the C-terminal, hPTH-(34-47), was inactive compared with an equimolar concentration of the fragment hPTH-(25-39), which stimulated both CK activity and DNA synthesis. These results in vivo are in line with previous findings in vitro; they provide further support for the suggestion that mid-region fragments of the PTH molecule could be used to induce bone formation without incurring the deleterious effect of bone resorption.

We have reported previously that parathyroid hormone (PTH) acts on cultured bone cells to stimulate creatine kinase (CK) activity and [3H]thymidine incorporation into DNA via phosphoinositide turnover, in addition to its other actions via increased cyclic AMP production. We also found that mid-region fragments of PTH stimulate [3H]thymidine incorporation into avian chondrocytes. In the present study of mammalian systems, we demonstrate differential effects of defined synthetic PTH fragments on CK activity and DNA synthesis, as compared with cyclic AMP production, in osteoblast-enriched embryonic rat calvaria cell cultures, in an osteoblast-like clone of rat osteosarcoma cells (ROS 17/2.8) and in chondroblasts from rat epiphysial cartilage cell cultures. Unlike full-length bovine (b)PTH-(1-84) or the fully effective shorter fragment human (h)PTH-(1-34), fragments lacking the N-terminal region of the hormone did not increase cyclic AMP formation, whereas they did stimulate increases in both DNA synthesis and CK activity. Moreover, the PTH fragment hPTH-(28-48) at 10 microM inhibited the increase in cyclic AMP caused by 10 nM-bPTH-(1-84). The increase of CK activity in ROS 17/2.8 cells caused by bPTH-(1-84) or hPTH-(28-48) was completely inhibited by either cycloheximide or actinomycin D, as was shown previously for rat calvaria cell cultures. These results indicated the presence of a functional domain of PTH in the central part of the molecule which exerts its mitogenic-related effects on osteoblast- and chondroblast-like cells in a cyclic AMP-independent manner. Since cyclic AMP formation by PTH leads to bone resorption, specific mid-region fragments of PTH might prove suitable for use in vivo to induce bone formation without concomitant resorption.

The stimulation of DNA synthesis in primary cell cultures of chicken chondrocytes by parathyroid hormone was studied by assaying [3H]thymidine incorporation into DNA. Optimal assay conditions were determined by varying cell age, plating density, and incubation time. Under these conditions DNA synthesis was significantly stimulated by parathyroid hormone (PTH) and some of its fragments: cells treated with human (h)PTH(1-84), bovine (b)PTH(1-34) and [Nle8,18,Tyr34]bPTH(3-34)amide and hPTH(13-34) displayed 2.6-fold enhanced [3H]thymidine incorporation in a dose-dependent manner. The fragment hPTH(28-48) led to a similar stimulation, whereas [Tyr43]hPTH(43-68) and [Tyr52,Asp76]hPTH(52-84) had no effect. Using a series of synthetic hPTH peptides covering the central region of the hormone molecule (residues 25-47), we could delimitate further this putative mitogenic functional domain to a core region between amino acid residues 30 and 34. The effect of PTH on [3H]thymidine incorporation could not be mimicked by forskolin, indicating that the corresponding signal is not mediated by cAMP. It is, however, inhibited by EGTA and cannot be provoked in the absence of calcium ions in the medium. Therefore, the results presented indicate a hitherto unidentified functional domain of PTH in the central part of the molecule which exerts its mitogenic effect on chondrocytes in a cAMP-independent manner but seems to involve calcium ions for signal transduction.