J. M. van der Meer

A two-receptor model for the action of parathyroid hormone on osteoblasts: A role for intracellular free calcium and cAMP

It has been suggested that intracellular Ca2+, in addition to cAMP, plays an important role in PTH-stimulated bone resorption. There is now strong evidence indicating that the osteoblast is the main target cell for PTH action, regulating indirectly, via cell-cell communication, osteoclastic bone resorption. In order to investigate the possible role of free cytosolic calcium in stimulated bone resorption, we studied the effects of the intact hormone (bPTH 1-84) and some of its fragments (bPTH (1-34), bPTH(3-34,) (Nle-8, Nle-18,Tyr-34) bPTH (3-34) amide) on their capacity to modify the cytosolic Ca2+ concentration in rat osteoblast-like cells. The experiments were performed using Quin-2, a fluorescent indicator of free calcium. We found an excellent correlation between the ability of PTH and PTH fragments to transiently increase cytosolic Ca2+ concentration in rat osteoblast-like cells and their ability to stimulate bone resorption in embryonic rat calvaria in vitro. On the other hand, no direct correlation was found for the cAMP and bone-resorbing responses. On the ground of these data we propose a two-receptor model for PTH action in osteoblasts, in which one receptor is coupled to the production of cAMP, whereas the other is involved in the increase of cytosolic Ca2+. Activation of both receptors by PTH (1-84) or PTH (1-34) leads to the full physiological response in osteoblasts, most probably the release of one or more factors which stimulate the activity of existing osteoclasts and others which stimulate the recruitment of additional osteoclasts.

Action of bPTH and bPTH fragments on embryonic bone in vitro: Dissociation of the cyclic AMP and bone resorbing response

SummaryThe effects of bPTH-(1-84), bPTH-(1-34), [Nle-8, Nle-18, Tyr-34] bPTH-(1-34), bPTH-(1-34) amide (NTA 1-34, desamino bPTH-(1-34), bPTH-(2-34), bPTH-(3-34), and [Nle-8, Nle-18, Tyr-34] bPTH-(3-34) amide (NTA 3-34) were tested in cultured bone cells, isolated from the osteoblast layers of fetal chicken calvaria (cyclic AMP) and in fetal rat calvaria (cyclic AMP, Ca release, and lactate production). Only bPTH-(1-84), bPTH-(1-34), and NTA 1-34 increased cyclic AMP production in a doserelated manner, both in calvaria and in bone cells, whereas all fragments (except NTA 3-34) stimulated bone resorption, the order of decreasing potency being bPTH-(1-84), NTA 1-34, bPTH-(1-34), desamino bPTH-(1-34), bPTH-(2-34), bPTH-(3-34). As in human cells, the antagonist NTA 3-34 inhibited specifically and in a dose-dependent way the cyclic AMP response of maximal concentrations of both bPTH-(1-84) and bPTH-(1-34) in rat calvaria and in chicken bone cells, when measured after short (15 min) and longer (1 1/2–16 h) incubation periods. In addition, measured after 4 h of incubation, NTA 3-34 completely inhibited bPTH-(1-84)-stimulated Ca release using maximal and submaximal concentrations. However, after 6–24 h of incubation, NTA 3-34 had no effect on bPTH-(1-84)-stimulated Ca and lactate release, even at an antagonist/agonist ratio up to 12.5 M, perhaps due to its lower affinity for the PTH receptor. From these findings we propose that (a) in bone there are two types of receptors, one governing demineralization via regulation of the calcium influx and one governing adenylate cyclase activity, and (b) the receptors are different from each other with respect to their affinities toward the agonists and the antagonist.

Different roles for calcium and cyclic AMP in the action of PTH: studies in bone explants and isolated bone cells.

In fetal mouse calvaria forskolin (0.1-100 microM), like PTH, stimulated cyclic AMP production in a dose-dependent way. The dose-response curve for forskolin-induced bone mineral release (24 hrs), however, demonstrated a biphasic character, showing stimulation at 0.1 microM and inhibition at 5 and 10 microM. In addition, forskolin-stimulated bone resorption reached a plateau after 48 hrs of incubation, a phenomenon which did not occur with PTH. Forskolin (0.1 microM) strongly stimulated PTH-induced cyclic AMP production in fetal mouse calvaria. However, PTH-stimulated bone resorption and PTH-induced increase in cytosolic free Ca2+ in bone fetal rat cells were not stimulated by forskolin (0.1 microM). 9-(Tetrahydro-2-furyl) adenine (100 microM) completely blunted PTH-stimulated cyclic AMP response in fetal mouse calvaria. PTH-stimulated bone resorption was also completely inhibited, but only after 6 hrs and not after 24 hrs of incubation. With nifedipine and varabamil PTH-stimulated bone resorption was significantly inhibited after 24 hrs of incubation and not significantly after 6 hrs of incubation. A23187 (1 microM) significantly stimulated PTH-stimulated cyclic AMP level and increased basal cytosolic free Ca2+ concentration in cultured rat bone cells. In calvaria, however, it had no effect on either basal and PTH-stimulated cyclic AMP production or on basal and PTH-stimulated bone resorption (6 and 24 hrs). From these observations it follows that in calvaria manipulation of intracellular cyclic AMP only (partially) affects bone resorption. This observation points to a role for an additional second messenger in establishing full blown bone resorption. Some of the results are published in short elsewhere.

Expression of the parathyroid hormone receptor and correlation with other osteoblastic parameters in fetal rat osteoblasts

Primary fetal rat calvarial cell cultures were examined for the expression of different osteoblastic parameters at the single cell level and in the whole population. The presence of the parathyroid hormone (PTH) receptor was studied by employing receptor autoradiography. After 3 days of culture, 10% of the cells expressed the PTH receptor. Immunolocalization of osteocalcin in 3-day-old cell cultures was found to be strongly correlated with the presence of the PTH receptor. Alkaline phosphatase (APase) localization in 3-day-old cultures correlated with only 69% of the PTH receptor expressing cells. Our results show that in 3-day-old rat calvarial cell cultures, only about 10% of the cells show markers of osteoblastic differentiation. The presence of the PTH receptor is strongly correlated with the presence of osteocalcin, but less with the presence of APase, indicating that it is the mature osteoblast that expresses the PTH receptor. After 7 days of culture, most receptor labeling, APase, and osteocalcin expression was found in multilayered areas of cells (nodules).

Heterogeneity of intracellular calcium responses to parathyroid hormone and thrombin in primary osteoblast-like cells and UMR106-01 cells: correlations with culture conditions, intracellular calcium concentration and differentiation state.

The present study evaluates the effect of parathyroid hormone (PTH) on intracellular calcium. Intracellular calcium ion concentrations ([Ca2+]i) in fetal rat osteoblasts in primary culture (ROB) and in UMR106-01 osteogenic sarcoma cells were monitored as changes in the ratio (R) of Fura-2 fluorescence intensities in single cells as well as populations of cells. In both single ROB and UMR106-01 cells, addition of 10(-7) M rat PTH1-34 and 3 NIH units/ml human thrombin resulted in heterogeneous responses in R values and therefore [Ca2+]i. PTH-induced calcium responsiveness of ROB was dependent on culture conditions, such that response frequencies were positively correlated with the percentage of fetal calf serum in the culture medium. PTH responsive ROB and UMR106-01 cells had significantly higher resting [Ca2+]i than unresponsive cells. PTH- or thrombin-mediated calcium signalling appeared not to be correlated to alkaline phosphatase activity in single ROB. Low percentages of cells responded to PTH in comparison to thrombin suggesting that an increase in [Ca2+]i is not a common PTH signalling pathway in osteoblasts in primary culture. Our data suggest that activation of this signalling pathway by PTH is culture condition dependent, possibly via a cell-cycle related increase in sensitivity of the pathway.

Non-radioactive in situ hybridization for the detection of cytomegalovirus infections

SummaryAcetylaminofluorene (AAF) modified cytomegalovirus (CMV) DNA probes have been applied for the rapid detection of CMV genomes by non-radioactive in situ hybridization in routinely obtained pathological material. To establish proper protocols, AAF modified mouse satellite DNA and mouse liver were used to investigate the procedural variables. Among these were type and time of fixation, glass slide coating for improved tissue adherence, protease permeabilization of sections, type and time of denaturation and hybridization, probe concentration, post-hybridization washing conditions and immunocytochemical detection. This research has led to a user-friendly procedure which, in addition to cells displaying a cytopathological effect typical for CMV infection detects with high sensitivity CMV carrying cells that show no histo-pathological alterations. It can be readily applied in routine clinical-diagnostic laboratories.

The effect of procedural and interactional criteria on procedural fairness judgments

In studying procedural fairness judgments, distinctions are made between (i) the mere presence of rules and procedures in the process of outcome allocation and dispute resolution, (ii) the application of these rules by a decision maker, and (iii) the enactment of procedures and rules in the interaction between a decision maker and involved parties. In line with Bies and Moag (1986), criteria that must satisfy the application of rules to be judged as fair are called procedural fairness criteria as distinguished from interactional fairness criteria. The hypothesis that interactional fairness criteria are more important in affecting fairness judgments than procedural fairness criteria is tested. Fifty-four subjects received information about a fictitious job application situation. The subjects judged the decision makers handling to the application procedure and his/her treatment of the applicant as fair or unfair. Three procedural and three interactional criteria and the final decision (hired or not) were used in the study. Results show that the decision makers consistent application of rules and his/her truthfulness to the applicant were judged as the most important factors in determining the fairness of the procedure. Accurate processing of information about the applicant and respectful treatment were judged as least important factors. Contrary to expectation, procedural criteria were judged on the average as equally important for determining fairness as interactional criteria. It is argued that the smaller than expected impact of the interactional criteria may be due to the fact that in the present study the entire application situation was evaluated and not the specific face-to-face aspects of the interaction. Results are in agreement with those of Tyler and Schuller (1990).

Differential depolarization-activated calcium responses in fetal and neonatal rat osteoblast-like cells

The present study evaluates differential occurrence of voltage-dependent calcium channels (VDCC) in the membranes of fetal (FROB) and neonatal (NROB) calvarian rat osteoblastic cells in primary culture. The intracellular calcium concentration ([Ca2+]i) was monitored upon depolarization of the cell membrane with the use of high K+ containing extracellular solutions. [Ca2+]i was measured in populations of cells as well as in individual cells using Fura-2, whereas the membrane potential (Em) was recorded in parallel experiments using patch-clamp techniques. Increasing the extracellular K+ concentration resulted in an instantaneous depolarization of Em of both FROB and NROB. This depolarization of Em did not significantly affect [Ca2+]i of populations of FROB and neonatal osteoblast precursors (NpROB). In contrast to FROB and NpROB, NROB populations responded to depolarization with significant transient [Ca2+]i increases that could be blocked by the calcium antagonist verapamil and were absent if extracellular Na+ was replaced for choline instead of K+. In individual cell measurements, response frequencies as well as the magnitude of [Ca2+]i responses upon depolarization of NROB were much higher than those of FROB, suggesting that more NROB than FROB possess VDCC. This phenomenon might point to a development-related expression of VDCC in the membranes of osteoblast-like cells.

Down-regulation and differential restoration of cAMP responses upon transient phorbol ester treatment of primary osteoblastic cells.

We studied cAMP responses induced by parathyroid hormone (PTH), prostaglandin E2 (PGE2) and forskolin in foetal rat calvariae-derived osteoblastic cells after 24 h treatment with a protein kinase C (PKC) activating phorbol ester. After this treatment, meant to down-regulate PKC activity, all tested cAMP responses were attenuated and were indeed accompanied by a decline in PKC activity. PTH receptor affinity was not altered and PTH receptor number was only slightly lowered after 24 h phorbol ester treatment. These results indicate that modulation of the cAMP responses by 24 h PMA treatment was mainly caused by a general impairment of adenylyl cyclase activity. Removal of the phorbol ester and subsequent culture for 2 days rendered the cells hyper-responsive to PTH: the PTH-induced cAMP response was 2 to 3 times higher than in control cells. Again no change in binding affinity of the PTH receptor was observed and receptor number was just 10% lower than in control cells. The PGE2- and forskolin-induced cAMP responses were not higher than normal. So, transient phorbol ester treatment leads to a differential, agonist-dependent restoration of the cAMP signalling system.

Individuals and laboratory populations

The basic strategy of the structured approach to population dynamics, as advocated in these notes, starts from mechanistic considerations on the individual level incorporating various amounts of detail about the underlying physiology. The resultingi-model is used as the substrate lor subsequent p- modelling. In the end we may say that we have explained populationphenomena from considerations on the i-level.