Age-dependent changes in myogenic precursor cell compartment sizes. Evidence for the existence of a stem cell

Individual myogenic cells were isolated from the pectoralis muscles of chick embryos from days 8-14 of embryogenesis. When separately cloned, these cells produced three types of colonies in culture: (1) Positive: all cells in the clone were terminally differentiated muscle cells; (2) negative: no cells in the clone were terminally differentiated muscle; (3) mixed: some cells in the clone were terminally differentiated muscle. Positive clones from all ages tended to contain 2n cells (n = 0, 1, 2, 3, 4). Negative clones were found in all sizes and did not cluster around powers of 2 in cell number. Mixed clones were, by far, the most common type among those clones larger than 24 in cell number. Estimates of cell numbers in embryonic muscle tissue revealed that, while the numbers of cells in all myogenic compartments increased steadily with embryonic age, the number and percentage of precursor cells that produced large mixed clones increased dramatically. Subclones, prepared from populations of cells equivalent to large mixed clones, yielded both small positive and large mixed colonies. This indicated that the precursors to the large mixed clones were also precursors to the smaller positive clones. These observations suggest a model for the myogenic lineage in which there exists a stem cell that can generate, by a series of asymmetric divisions, cohorts of terminally differentiated muscle cells. The model can explain the asynchrony of production of terminally differentiated muscle cells both in vitro and in vivo.     

Effects of transforming growth factor-beta on normal clonal bone cell populations.

Although transforming growth factor-beta (TGF-beta) has been implicated in the local regulation of bone growth and remodelling, its specific effects on different subpopulations of bone cells have not been elucidated. Cells derived from bone are known to be heterogeneous and include both cells of different lineages and osteoblastic populations with different levels of expression of osteoblast-associated properties. Consequently, we have isolated clonal populations of bone cells to examine more precisely the effects of TGF-beta on individual subpopulations. Several clonal populations were isolated by limiting dilution from cells derived from 21-day-old fetal rat calvaria. Two of these clones, RCA 11 and RCB 2, were used here. While the two clones responded similarly to parathyroid hormone (PTH) and isoproterenol (ISP) with increases in intracellular cAMP, prostaglandin E2 (PGE2) elicited a 10-fold higher response in RCB 2 cells compared with RCA 11. RCB 2 cells expressed a 10-fold higher alkaline phosphatase activity compared with RCA 11. Both clones synthesized a variety of bone matrix associated proteins, but only RCA 11 synthesized SPP-1 (osteopontin) constitutively. TGF-beta stimulated growth of RCB 2 cells after 24 and 48 h of treatment, but had no effect on growth of RCA 11. TGF-beta supported anchorage-independent growth of RCB 2 cells, but not that of RCA 11. A 24-h exposure to TGF-beta decreased cAMP responsiveness to PTH and ISP slightly in both clones, but had no effect on PGE2 responses. Significant reductions in alkaline phosphatase activity were seen in both clones after 24- and 48-h treatments with TGF-beta.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recognition of Rous sarcoma virus-induced tumor antigens by cytotoxic T lymphocytes (CTL): studies on specificity of killing by CTL employing H-2 congenic and recombinant mouse tumor cells

The specificities of cytotoxic T lymphocytes (CTL) were studied for the analysis of CTL against tumor-specific cell surface antigen(s) (TSSA) of non-virus-producing tumor cells induced by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) in B10 congenic and recombinant mice. Eight CTL clones were established from immune spleen cells of B10.A(5R) mice. These clones demonstrated six patterns of cytotoxic reactivity in vitro: Two clones showed H-2 restriction in tumor cell lysis. Two other clones had the capacity to lyse syngeneic, H-2K-compatible B10 and H-2-incompatible B10.A(4R) tumor cells, but not YAC-1 cells. One clone had cytotoxic activity against syngeneic, H-2D-compatible B10.D2 tumor cells and YAC-1 cells, but not against H-2-incompatible tumor cells. One clone had cytotoxic activity against syngeneic and YAC-1 tumor cells, but not against either H-2-compatible or H-2-incompatible tumor cells. One clone had lytic activity to syngeneic, H-2-compatible, H-2-incompatible, and YAC-1 tumor cells. Another clone killed H-2-incompatible B10.A(4R) tumor and YAC-1 cells, but not syngeneic or H-2-compatible tumor cells. All these clones strongly expressed surface Thy-1.2 antigens, whereas the expression of Lyt-1.2 and Lyt-2.2 antigens was different from clone to clone. These results demonstrate heterogeneity of both lytic specificity and phenotype of CTL against RSV-induced mouse tumor cells, suggesting the existence of multiple antigenic sites on the RSV TSSA recognized by CTL populations.     

Natural killer cell clones can efficiently process and present protein antigens.

NK cell clones obtained from three different donors were tested for their ability to present soluble proteins to Ag-specific T cell clones. All NK clones were CD2+CD3-CD56+, whereas the expression of CD16 varied from clone to clone. The NK cell clones were able to process and present tetanus toxoid (TT) to TT-specific T cell clones in a class II HLA restricted manner. The capacity of NK cell clones to function as APC was also observed using the house dust mite allergen Der p I and the Der p I-derived peptide Val89-Cys117. As with EBV-transformed B cell line, NK cell clones could present the peptide 3-13 derived from the 65-kDa heat shock protein of Mycobacterium leprae, but they were unable to present the whole M. leprae Ag. Freshly isolated NK cells, IL-2-activated NK cells, and NK cell lines expanded in vitro could also process and present TT. The ability of the different NK populations to act as accessory cells correlated with their levels of class II HLA expression. These data demonstrate that NK cell clones can efficiently function as APC, however they may be restricted in the types of Ag that they can process.     

Cloned auto-Ia-reactive T cells elicit lichen planus-like lesion in the skin of syngeneic mice

To explore the physiologic or pathologic roles of autoreactive T cells, we examined immunological functions of several autoreactive mouse T cell clones in vitro and in vivo. All of the T cell clones were Lyt-2-, L3T4+ and showed self-I region-restricted proliferative responses (one clone was self-I-E restricted, the other clones were self-I-A restricted). One clone derived from C57BL/6 mouse and reactive to the self-I-Ab product (clone bb1-2) showed cross-reactivity to the I-Ak product. Among four such auto-Ia-reactive T cell clones examined, one clone produced fairly large amounts of interleukin 2 (IL 2) in response to syngeneic stimulator cells, and mediated help for the in vitro cytotoxic T cell (CTL) responses of syngeneic thymocytes, whereas this clone did not mediate in vitro antibody responses of syngeneic B cells. The other three clones were producers of small amounts of IL 2 and did not mediate the in vitro CTL responses. Among the three clones, clone bb1-2 showed strong regulatory function, and clone kk-1 (B10.BR origin and self-I-Ak reactive) showed weak regulatory function in vitro antibody responses of syngeneic B cells. The physiologic or pathologic roles of autoreactive T cells in vivo were explored by injecting subcutaneously clone kk-1 T cells or clone bb1-2 T cells into the footpads of the respective syngeneic mice. Clone kk-1 T cells injected into syngeneic mice elicited swelling of the footpad and marked accumulation of mononuclear cells in the dermis, leaving the epidermis intact, as in the delayed-type hypersensitivity reaction. As a notable finding, clone bb1-2 T cells injected into syngeneic mice elicited marked swelling of the footpad and lichen planus-like skin lesions, i.e., infiltration of lymphocytes in the epidermis and epidermal cell damage. The lymphocytes infiltrating in the epidermis were evaluated, as were the injected clone bb1-2 T cells expressing the Lyt-1.2 phenotype, by examination of the skin lesions elicited in C3H/He mice (H-2k, Lyt-1.1, 2.1) by the clone T cells. Clone bb1-2 T cells exerted in vitro cytotoxicity against H-2b and H-2k target cells, whereas clone kk-1 T cells did not show any cytotoxic activity, indicating a correlation between the cytotoxic activity of clone bb1-2 T cells and their ability to elicit lichen planus-like lesions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Delayed malignancy and altered growth properties of somatic cell hybrids between rat hepatoma and mouse L-cells.

Somatic cell hybrids of mouse L-cells with rat HTC cells were studied for their growth properties in vitro and tumorigenicity in vivo. Three hybrid clones were selected for detailed study. All were delayed in tumor formation in nude mice compared with the parent lines despite their varied growth properties in vitro. One clone was sensitive to density dependent inhibition of growth (DDIG) and had a relatively low saturation density. A second clone was not sensitive to DDIG and had a higher saturation density. The third clone had atypical morphology, was insensitive to DDIG, and had a relatively high saturation density. All of the clones studied produced colonies suspended in agarose gel which were much smaller than those of the parents incubated for the same period of time. Only the pattern of growth in agarose gel corresponded to the delayed tumor formation in vivo of the hybrids. Sensitivity to DDIG and saturation density were not consistent with tumor growth. The hybrid clone that was sensitive to DDIG was the only one of the three that had a nearly complete set of chromosomes derived from each of the parents. The chromosome numbers of all three clones were unchanged after growth in agarose or as tumors in the nude mice.     

Fluorescence activated cell sorting reveals heterogeneous and cell non-autonomous osteoprogenitor differentiation in fetal rat calvaria cell populations

Identification of osteoblast progenitors, with defined developmental capacity, would facilitate studies on a variety of parameters of bone development. We used expression of alkaline phosphatase (ALP) and the parathyroid hormone/parathyroid hormone-related protein receptor (PTH1R) as osteoblast markers in dual-color fluorescence activated cell sorting (FACS) to fractionate rat calvaria (RC) cells into ALP(-)PTH1R(-), ALP(+)PTH1R(-), ALP(-)PTH1R(+), and ALP(+)PTH1R(+) populations. These fractionated populations were seeded clonally (n = 96) or over a range of cell densities ( approximately 150-8,500 cell/cm(2); n = 3). Our results indicate that colony forming unit-osteoblast (CFU-O)/bone nodule-forming cells are found in all fractions, but the frequency of CFU-O and total mineralized area is different across fractions. Analysis of these differences suggests that ALP(-)PTH1R(-), ALP(-)PTH1R(+), ALP(+)PTH1R(-), and ALP(+)PTH1R(+) cell populations are separated in order of increasing bone formation capacity. Dexamethasone (dex) differentially increased the CFU-O number in the four fractions, with the largest stimulation in the ALP(-) cell populations. However, there was no significant difference in the number or size distribution of CFU-F (fibroblast) colonies that formed in vehicle versus dex. Finally, both cell autonomous and cell non-autonomous (i.e., inhibitory/stimulatory effects of cell neighbors) differentiation of osteoprogenitors was seen. Only the ALP(-)PTH1R(-) population was capable of forming nodules at the clonal level, at approximately 3- or 12-times the predicted frequency of unfractionated populations in dex or vehicle, respectively. These data suggest that osteoprogenitors can be significantly enriched by fractionation of RC populations, that assay conditions modify the osteoprogenitor frequencies observed and that fractionation of osteogenic populations is useful for interrogation of their developmental status and osteogenic capacity.     

Comparison of two host cell range variants of feline immunodeficiency virus.

Two molecular clones of feline immunodeficiency virus were compared. The first clone, 34TF10, was from a Petaluma, Calif., isolate; the second, PPR, was isolated from a cat in the San Diego, Calif., area. The cats from which the isolates were obtained suffered from chronic debilitating illnesses. The two molecular clones differed in their in vitro host cell range. The 34TF10 clone infected the Crandall feline kidney and G355-5 cell lines, but replicated less efficiently on feline peripheral blood leukocytes. In contrast, the PPR clone productively infected the primary feline peripheral blood leukocytes but not Crandall feline kidney or G355-5 cells. The 34TF10 and PPR clones had an overall sequence identity of 91%. The env gene was the least conserved (85% at the amino acid level). Additionally, the potential open reading frame for a Tat-like protein, ORF 2, contained a stop codon in the 34TF10 isolate which was not found in the PPR clone. This truncation did not prevent in vitro or in vivo replication of 34TF10. Two splice acceptor sites were identified in the 34TF10 clone. One was 5' to the beginning of the putative tat open reading frame, and the other was 5' to the putative vif product. Both of these acceptor sites were conserved in the PPR clone. The long terminal repeats of the viruses were 7% divergent between the two clones, with a lack of conservation in putative NF-kappa B, LBP-1, and CCAAT enhancer-promoter sites.     

Anti-idiotypic antibodies against UV-induced tumor-specific CTL clones. Preparation in syngeneic combination

In this study, we first established several CTL clones of (BALB/c x C57BL/6)F1 origin that were specific for either syngeneic UV female 1 or UV male 1 fibrosarcoma cell lines. All the CTL clones had Thy-1+ Lyt-2+ L3T4- phenotypes and showed Kd restriction when lysing the corresponding target cells. Sera obtained from syngeneic animals immunized with three CTL clones, 10B-5 for UV female 1, and CTL9 and CTL10 for UV male 1, showed specific inhibition of target cell lysis with the corresponding CTL clones. The inhibitory activities were found in sera of the majority of immunized animals. Because the inhibitory activity resides in protein A-binding fraction, mAb were produced by hybridizing spleen cells of hyperimmune animals. N1-56 was thus obtained from a mouse immunized with 10B-5 CTL clone reactive with UV female 1. N1-56 was clonotype specific, reacting with 10B-5 but not with other CTL lines or leukemia cell lines. No N1-56+ cells were detectable in thymocytes, lymph node cells, or spleen cells of either naive or UV female 1-immune CB6F1 mice. Immunoprecipitation showed that N1-56 reacts with 90,000 Mr molecules on 10B-5 CTL clone under nonreducing conditions and 45,000 Mr molecules under reducing conditions, indicating its reactivities with idiotypic determinants of TCR on the CTL clone. N1-56 inhibited lytic activity of 10B-5, but neither N1-56 nor alpha-10B-5 hyperimmune serum inhibited that of alpha-UV female 1 mixed lymphocyte tumor cell culture cells. N1-56 induced proliferation of 10B-5 without addition of Ag.     

Marrow stroma-derived osteogenic clonal cell lines: Putative stages in osteoblastic differentiation

This report documents characterization of five osteogenic cell subpopulations of bone marrow stroma. The clonally derived cell lines were isolated from the parental line MBA-15 known to express osteoblastic-associated features in vitro and to form bone in vivo. The latter, presumably “arrested” at a particular stage along the osteogenic lineage, are useful models to study the processes involved in the differentiation of bone forming cells. The clones differ in their morphology, proliferation rate, quantities and distribution of extracellular matrix proteins, levels of alkaline phosphatase activity and activation of adenylate cyclase by parathyroid hormone and/or prostaglandin E. These properties have been retained during prolonged growth and subculturing through many passages. MBA-15.4 is a presumptive preosteoblast with a fibroblast-like appearance; it proliferates rapidly, synthesizes equal amounts of collagen and noncollagenous proteins, and produces constitutively low levels of alkaline phosphatase. This clone has PGE₂-stimulated adenylate cyclase activity and a very low constitutive response to PTH. On the other hand, MBA-15.6 has a large polygonal morphology with limited proliferative potential, synthesizes twice as much noncollagenous proteins as collagen, has high alkaline phosphatase activity, and responds strongly to PTH. The characteristics of the other clones place them between these two categories. The effects of 10^?7 M dexamethasone or 10^?12–10^?8 M 1,25 dihydroxyvitamin D₃ on growth and differentiation further strengthen the variance between these clones. The different in vitro characteristics of the various clones were directly reflected in their bone formation ability in vivo. When transplanted under the renal capsule, MBA-15.33 formed a thick fibrous tissue, MBA-15.4 formed small foci of bone, and MBA-15.6 formed massive woven bone at the same period of time. © 1993 Wiley-Liss, Inc.     

Rapidly expanded activated human killer cell clones have strong antitumor cell activity and have the surface phenotype of either T gamma, T-non-gamma, or null cells

Cloned lymphoid cell lines showing cytolytic activity were derived from natural killer (NK) cell-enriched cell fractions obtained by fluorescence-activated cell sorting of cells that reacted with B73 .1, an NK cell-specific monoclonal antibody (MCA). The clones were cultured for more than 30 generations (i.e., more than 10(9) descendants from a single cell). The rapid expansion was achieved by using a special culture system developed for this purpose and based on the use of two types of allogeneic feeder cells. Three phenotypically different types of cytotoxic clones were obtained. These clones showed a broad spectrum of cytolytic activity against several NK-susceptible and NK-nonsusceptible tumor target cells. One of these clones had the following binding pattern to MCA: B73 .1+, T3-, T4-, T8-, HNK1 -, and Lyt-3-. These cells formed rosettes with IgG-coated erythrocytes but not with sheep erythrocytes, and therefore might be null cell-derived. Most of the cytotoxic clones showed the following phenotype: B73 .1+, T3-, T4-, T8-, HNK1 -, Lyt-3+, E+, and EA-gamma +. These clones were probably derived from T-gamma cells. In addition, one clone with cytolytic activity was derived from B73 .1- cells. This had the phenotype B73 .1-, T3+, T4-, T8-, HNK1 -, Lyt-3+, E+, and EA-gamma-, and may be of T-non-gamma cell origin. About 10 noncytolytic clones showed the phenotype B73 .1-, T3+, T4, or T8+, HNK1 -, Lyt-3+, Ia+, E+, and EA-gamma -. An absolute correlation was found between the presence of the B73 .1 antigen, the absence of the T3 marker, and the capacity of the cells to form EA rosettes. Furthermore, all clones except one (Lyt-3-) formed E rosettes. Although the in vitro life span varied from clone to clone, B73 .1- clones generally grew faster and for longer times (greater than or equal to 50 generations) than did B73 .1+ ones (less than or equal to 40 generations). The cytolytic activity, cell surface phenotype as determined with MCA, rosette formation, and target cell specificity spectrum remained stable over the entire culture period. We conclude that the majority of the activated MHC-nonrestricted cytolytic clones obtained in this culture system show a particular phenotype. These cells can be expanded to large numbers. Whether or not these clones might be derived from B73 .1+, HNK1 + NK cells with the morphologic appearance of large granular lymphocytes will be discussed.     

Ontogeny-derived hematopoietic stem cells divide successively producing clones of differentiated hematopoietic cells

Forty seven individual haemopoietic cell clones bearing unique radiation markers were studied in long-term bone marrow cultures. Throughout cultivation, clones appeared at different times, 1 to 12 weeks after explanation, survived during 1-10 weeks and markedly varied in size. Usually, the number of metaphases characteristic of an individual clone rapidly increased, achieved a maximum and declined. The cells of disappeared clones were never seen again. The experimental results provide further evidence for the model of haemopoiesis by clonal succession. The data obtained are discussed with respect to the functioning of haemopoietic stem cell population.     

Cytoplasmic microtubules in transformed mouse x nontransformed human cell hybrids: correlation with in vitro growth.

Patterns of cytoplasmic microtubules in somatic cell hybrids between transformed mouse cells and nontransformed human skin fibroblasts were examined using antitubulin antibodies as an immunofluorescent probe. Nontransformed cells have been shown to exhibit an extensive cytoplasmic microtubule complex (CMTC), while in transformed cells, this complex is greatly diminished. The hybrid populations contained both types of cells. In addition, they contained cells with previously undescribed intermediate CMTC phenotypes. The percentage of each phenotype present in hybrid populations was determined for sixteen hybrid clones. Seven clones were found which appeared transformed on the basis of their CMTC pattern. The others were comprised of various proportions of all the cell types described. Repeated quantitation of the proportions of these types in the hybrid populations showed them to be stable with time in culture. Growth in vitro of the hybrid clones was assayed by determining their saturation densities, their plating efficiencies on plastic and their colony-forming abilities in soft agar. In vitro growth of a cell population was found to be directly proportional to the percentage of cells in the population which showed the greatly diminished CMTC pattern which has been described for transformed cells. This is strong evidence that a greatly reduced CMTC is associated with transformed behavior, especially the increased capacity of transformed cells for in vitro growth.     

Characterization of human T-lymphocyte clones (TLCs) specific for HLA-region gene products

Clones of lymphocytes, primed in vitro to HLA-DR1; Dw1, were tested for allospecific proliferation on a panel of thirty-one HLA-phenotyped stimulating cells. No clone was restimulated exclusively by cells sharing the DR1; Dw1 priming antigens and most clones were restimulated by subsets of cells bearing DR1; Dw1. Generally, positive responses were at least 20-fold higher than autologous negative controls. Peak proliferative responses occurred around 72 h and varied, depending on the stimulating cell as well as the responding clone. Selected clones were induced to proliferate only by cells incapable of forming rosettes with sheep erythrocytes. Specific proliferation by TLCs was blocked by monoclonal DR-specific antibodies, but not by monoclonal anti-Thy 1.2. Genetic studies demonstrated that TLCs detected some cell-surface determinants that are encoded by genes in linkage disequilibrium with HLA and others that may not be linked to the human major histocompatibility complex.Abbreviations used in this paper ³HTdR tritiated methylthymidine - HTC homozygous typing cell - MHC major histocompatibility complex - HLA human MHC - MLC mixed leukocyte culture - PBL peripheral blood lymphocytes - PLT primed lymphocyte typing - TCGF T-cell growth factor - IL-2 interleukin 2 - TLC T-lymphocyte close     

Stimulation by defined parathyroid hormone fragments of cell proliferation in skeletal-derived cell cultures.

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.     

Comparison of the Frequencies of Spontaneous and Chemically-Induced 5-Bromodeoxyuridine-Resistance Mutations in Wild-Type and Revertant Bhk-21/13 Cells

5-bromodeoxyuridine resistance mutations induced by mutagenesis were studied. The average expression time for induced mutations varied with the concentration of the mutagen ethyl methanesulfonate (EMS). However, a constant number of two generation times was necessary for half maximal expression of induced mutations. Also, induced mutation rates were compared under optimal expression conditions for bromodeoxyuridine, fluorodeoxyuridine and azaguanine resistance markers. Ten independent bromodeoxy-uridine-resistant clones were tested for reversion. Two clones reverted-one spontaneously and the other after mutagenesis. The spontaneous rate of mutation to bromodeoxyuridine resistance, estimated by the fluctuation test, was high in revertant clones (4 x 10(-6) / cell / generation) and low in the wild-type cells (< 3.5 x 10(-8) / cell / generation). A comparison of induced mutation frequencies at variable EMS concentrations showed a single-hit curve for revertant clones and a multihit curve for the wild-type cells. Thymidine kinase activities of resistant clones were usually less than 2% of that of the wild-type clone. Inducibility, thermal stability and intracellular localization of the thymidine kinases of the wild-type cells and of a revertant clone were identical. A low, but significant (P < 0.10), Km discrepancy was observed between enzyme extracts of these lines. The genetic implications of these results are discussed.     

Electrophysiological differences between bone cell clones: membrane potential responses to parathyroid hormone and correlation with the cAMP response

Electrophysiological measurements on three clonally derived bone cell populations showed a positive correlation between longer-term hyperpolarizing membrane potential responses to parathyroid hormone (PTH) and an intracellular cAMP response to PTH. One clone (RCJ 1.20) had no sustained electrophysiological response and no cAMP response to PTH. Another clone (ROS 17/2.8) had both a sustained hyperpolarizing response and a cAMP response to PTH. The third clone (RCB 2.2) initially had both an electrophysiological response and a cAMP response to PTH, but both responses were lost after prolonged growth in culture. Application of dibutyryl cAMP to RCJ 1.20 and ROS 17/2.8 cells produced both transient and sustained hyperpolarizing responses. Application of isobutylmethylxanthine produced a sustained hyperpolarization. These results suggest that the hyperpolarizing response to PTH is related to a cAMP-mediated increase in Ca2+ conductance, which leads to an increase in Ca2+-activated K+ conductance. The pronounced membrane potential spikes and fluctuations that occur in some of the clonal lines were shown to be unrelated to the hyperpolarizing response to PTH. This was demonstrated by the lack of correlation between the occurrence of the spikes or fluctuations and the occurrence of the hyperpolarizing response to PTH in the various cell lines, by the lack of effect of PTH on the spikes and fluctuations, and by the lack of effect on the hyperpolarizing response to PTH of verapamil and quinine, both of which significantly reduce the spikes and fluctuations.