Identification of connexin43 (alpha1) gap junction gene mutations in patients with hypoplastic left heart syndrome by denaturing gradient gel electrophoresis (DGGE).

Gap junction channels formed by the connexin43 protein are considered to play crucial roles in development and function because they allow the direct cell-to-cell exchange of molecules that mediate multiple signaling events. Previous results have shown that connexin43 channels are intricately gated by phosphorylation and that disruption of this regulation gives rise to severe heart malformations and defects of laterality in human, chick and frog. Here we report the identification of connexin43 gene mutations that represent a minor population of connexin43 alleles, which could be reliably detected by using denaturing gradient gel electrophoresis (DGGE) to visualize normal and mutant DNAs that were separately sequenced. In contrast, sequencing of total PCR products without DGGE-pre-selection failed to consistently identify these mutations. Forty-six controls and 20 heart transplant recipients were examined in this study. In the latter group, 14 children had hypoplastic left heart syndrome (HLHS) in which connexin43 gene defects were detected in eight. The remaining six transplant patients with HLHS and all controls showed no defects. All eight HLHS children with gene defects had the same four substitutions: two that were silent polymorphisms, and two that were missense, replacing arginine codons at positions 362 and 376 with codons for glutamines. All four of these substitutions are identical to the nucleotide sequence of the connexin43 pseudogene, suggesting the possibility of an illicit recombination. A breakpoint region was identified 5' to the mutation site in a 63bp domain that is 100% identical in the gene and pseudogene. Results from in vitro phosphorylation indicate that the absence of arginines 362 and 376 completely abolishes phosphorylation in the connexin43 channel regulation domain suggesting a possible mechanism for the pathologies associated with HLHS.

N-acetylneuraminic acid (NANA) stimulates in situ cyclic AMP production in tentacles of sea anemone (Aiptasia pallida): possible role in chemosensitization of nematocyst discharge.

Cnidocytes, the stinging cells of cnidarians, optimally discharge nematocysts in response to combined physical contact and stimulation of specific chemoreceptors. In the tentacles of certain sea anemones, the primary chemoreceptors bind N-acetylated sugars, such as N-acetylneuraminic acid (NANA). Sensitization with NANA predisposes contact-sensitive mechanoreceptors (CSMs) to trigger discharge in response to physical contact. In the ectoderm of sea anemone tentacles, cnidocyte/supporting cell complexes (CSCCs) control and trigger nematocyst discharge. Previous findings have implicated cyclic AMP (cAMP) as a second messenger in NANA-sensitized nematocyst discharge. However, no reports have directly demonstrated that the cAMP content of tentacles changes in response to NANA stimulation. We now show that NANA elevates in situ cAMP levels in a dose-dependent manner in the ectoderm of tentacles from the sea anemone Aiptasia pallida. However, the endoderm of tentacles shows no detectable cAMP response to NANA. The effect of NANA on the cAMP content of the ectoderm is biphasic. Micromolar NANA increases the in situ cAMP level, with a maximal response occurring at 1.8x10(-5)mol x l(-1) NANA. At higher NANA concentrations, the cAMP content decreases to that of controls. Because the cAMP dose/response curve to NANA coincides precisely with the dose/response curves of NANA-sensitized nematocyst discharge and nematocyst-mediated adhesive force, a second-messenger role for cAMP in NANA-sensitized nematocyst discharge is strongly suggested. The addition of isobutyl-1-methylxanthine (IBMX) to the medium with sea anemones increases tissue cAMP levels both in the absence and in the presence of NANA. However, anesthetizing anemones in sea water containing high levels of Mg(2+) blocks the NANA-stimulated cAMP response of the ectoderm. In addition, our results suggest that NANA-stimulated cAMP may activate endogenous cAMP-dependent protein kinase (PKA) in broken cell preparations of tentacles. Thus, NANA-stimulated cAMP may function as a second messenger in the NANA chemosensory signaling pathway controlling nematocyst discharge.

Misregulation of connexin43 gap junction channels and congenital heart defects.

Although there is general agreement that gap junction channels formed by the connexin43 (Cx43; alpha 1) protein most likely have important roles during heart development, evidence to support this view has been equivocal. Lacking this information, it is difficult to understand the basis of heart malformations found in the Cx43 knockout mice and in children with a severe form of visceroatrial heterotaxia that coincides with missense mutations of the Cx43 gene. To address this issue we used a combination of western blots to follow the emergence of Cx43 in heart, and in vitro and in vivo phosphorylation to assess the effect of mutation on Cx43 phosphorylation. We evaluated the activity ratios of cAMP-dependent protein kinase and protein kinase C in hearts of 8.5-day-old mouse embryos through to birth. The results demonstrate that Cx43 is present in the native phosphorylated species in day 8.5 hearts and thereafter. Further, the activities of cAMP-dependent protein kinase and protein kinase C are mirror images of each other during the 8.5-10.5 days of early heart development. From these results we conclude that Cx43 gap junction channels are present and capable of being regulated by day 8.5 of embryonic heart development.

Effect of leukotriene inhibitor on salicylate induced morphologic changes of isolated cochlear outer hair cells.

Our previous studies have shown that salicylate ototoxicity is associated with decreased levels of prostaglandins (PGs) and increased levels of leukotrienes (LTs) in the perilymph. Other studies have demonstrated that salicylate ototoxicity is associated with decreased cochlear blood flow, reversible changes in isolated cochlear outer hair cells (OHCs), and decreased otoacoustic emission. We have shown that pretreatment with an LT inhibitor prevents salicylate induced hearing loss, a decrease in cochlear blood flow and changes in otoacoustic emissions. The objectives of the current study were to determine the effect of exposure of salicylate and LTs on the morphology of isolated OHSc and to determine the effect of LT inhibitors on salicylate induced morphologic changes of isolated OHCs. Isolated OHCs from chinchilla cochlea were exposed to different test solutions. The groups included sodium salicylate (10 mM) with or without pretreatment with an LT inhibitor (L-663, 536, 30 microM), 0.1 or 1.0 microM solution of LTC4, LTD4, LTE4, and two control solutions, standard bathing solution (SBS) or leukotriene inhibitor alone. Osomolality of all solutions were kept at 305 +/- 5 mmolkg-1. The OHCs were observed under an inverted microscope. Images were stored onto a computer and analyzed later. OHCs exposed to the salicyalate developed a decrease in mean cell length. The exposure of OHCs to LTC4, LTD4, and LTE4 also demonstrated a similar decrease in mean cell length. Cells in the control SBS or LT inhibitor alone groups did not show any change. OHCs exposed to salicylate in the presence of the LT inhibitor did not exhibit morphologic changes. This study suggest that arachidonic acid metabolites, especially an increase in the concentration of LTs, seem to play an important role in the pathogenesis of salicylate ototoxicity.

Regulation of cAMP-dependent protein kinase: enzyme activation without dissociation.

It has become axiomatic that, in contrast to other protein kinases, cAMP-dependent protein kinase (cAPK) is activated only when its catalytic (C) and regulatory (RII2) subunits dissociate. To directly evaluate this postulation, the ability of cAMP to dissociate the holoenzyme form of cAPK was examined by measuring the rotational mobility of the carboxyfluorescein-labeled C subunit (CFC) complexed to the dimeric RII2 regulatory subunit under equilibrium conditions. The rotational mobility was determined from an analysis of the time-resolved emission anisotropy of the CFC subunit. The time-resolved anisotropy decays were best fitted by a sum of two exponentials for both the free CFC subunit and the RII2CFC2 complex (holoenzyme). In the absence of cAMP, the two rotational correlation times (phi F and phi S) were 1.7 +/- 0.3 and 18.3 +/- 0.7 ns for the free CFC subunit and 2.3 +/- 0.1 and 93 +/- 2 ns for the RII2CFC2 complex, respectively. The faster rotational correlation times can be attributed to the localized rotations of the label and the slower rotational correlation times to the global rotations of the entire molecule. The addition of cAMP had no significant effect on either the fast or the slow rotational correlation time of the RII2CFC2 complex (phi F = 2.0 +/- 0.2 ns and phi S = 93 +/- 9 ns). Control experiments established that the RII2CFC2 complex was fully activated by cAMP at the same concentrations (0.2-0.4 microM) used for the anisotropy measurements. Together, the results demonstrate (1) that cAMP can induce the catalytic activity of cAPK without subunit dissociation and (2) that cAMP binding to holoenzyme is insufficient to explain its in vivo dissociation.

Mutations of the Connexin43 gap-junction gene in patients with heart malformations and defects of laterality.

BACKGROUND: Gap junctions are thought to have a crucial role in the synchronized contraction of the heart and in embryonic development. Connexin43, the major protein of gap junctions in the heart, is targeted by several protein kinases that regulate myocardial cell-cell coupling. We hypothesized that mutations altering sites critical to this regulation would lead to functional or developmental abnormalities of the heart. METHODS: Connexin43 DNA from 25 normal subjects and 30 children with a variety of congenital heart diseases was amplified by the polymerase chain reaction and sequenced. Mutant DNA was expressed in cell culture and examined for its effect on the regulation of cell-cell communication. RESULTS: The 25 normal subjects and 23 of the 30 children with heart disease had no amino acid substitutions in connexin43. All six children with syndromes that included complex heart malformations had substitutions of one or more phosphorylatable serine or threonine residues. Four of these children had two independent mutations, suggesting an autosomal recessive disorder. Five of these children had substitutions of proline for serine at position 364. A seventh child, with a different heart condition, also had a point mutation in connexin43. Transfected cells expressing the Ser364Pro mutant connexin43 sequence showed abnormalities in the regulation of cell-cell communication, as compared with cells expressing normal connexin43. CONCLUSIONS: Mutations in the connexin43 gap-junction gene, which lead to abnormally regulated cell-cell communication, are associated with visceroatrial heterotaxia.

Regulation of cell-cell communication mediated by connexin 43 in rabbit myometrial cells.

The aim of this study was to evaluate the rapid regulation of cell-cell communication by using the microinjection of purified cAMP-dependent protein kinase (protein kinase A), the Ca(2+)-phospholipid-dependent protein kinase (protein kinase C), or the inhibitor proteins (PKI and CKI) that are, respectively, specific for each of these enzymes. Gap junction phenotypes of myometrial tissue and cells were studied by means of immunocytochemistry with antibody to connexin 43 (alpha 1; Cx43). Cells were enzymatically disaggregated from myometrium of nonpregnant, mid-pregnant (Day 14), and late-pregnant (Day 29) rabbit uteri (n = 8 per group) and seeded at high density such that after 4 days, cultures had the appearance of a cross-sectioned myometrium. Purified proteins and their subunits were microinjected, and intercellular communication was evaluated by monitoring Lucifer Yellow dye transfer. Cultures were treated with 0.5 mM 8Br-cAMP (8-bromo adenosine 3',5' cyclic monophosphate) or 10 microM OAG (1-oleoyl-2-acetyl-sn-glycerol), which, respectively, activate protein kinase A and protein kinase C. Immunoreactive Cx43 and cell-cell communication were examined 5 min to 2 h later. Cx43 was detected in myometrial cryosections and cultured cells by indirect immunofluorescence, and its expression increased with gestation. Exposure to 8Br-cAMP increased the amount of immunoreactive Cx43. Basal dye transfer was minimal in nonpregnant cells, increased in cells of mid-pregnant uteri, and was maximal in late-pregnant cells. Treatment with 8Br-cAMP enhanced transfer in mid- and late-pregnant cells but had no obvious effect on cells from nonpregnant animals. OAG treatment inhibited dye transfer in greater than 95% of the cells tested irrespective of pregnancy status. PKI inhibited cell-cell communication within 2 min and up to 40 min. Injection of free catalytic subunit of protein kinase A following PKI inhibition restored communication within 2-3 min, with maximal transfer in 4-5 min. Protein kinase C inhibited communication, which resumed in < 3 min after injection of CKI. We conclude that rabbit myometrial cells engage in Cx43-mediated cell-cell communication and that this process increases during pregnancy. Further, activators of protein kinase A or injected free catalytic subunit rapidly enhances cell-cell communication, whereas activators of protein kinase C or the enzyme itself diminishes this process.

In situ regulation of cell-cell communication by the cAMP-dependent protein kinase and protein kinase C.

The effects of cAMP-dependent protein kinase A and protein kinase C on cell-cell communication have been examined in primary ovarian granulosa cells microinjected with purified components of these two regulatory cascades. These cells possess connexin43 (alpha 1)-type gap junctions, and are well-coupled electrotonically and as judged by the cell-to-cell transfer of fluorescent dye. Within 2-3 min after injection of the protein kinase A inhibitor (PKI) communication was sharply reduced or ceased, but resumed in about 3 min with the injection of the protein kinase A catalytic subunit. A similar resumption also occurred in PKI-injected cells after exposure to follicle stimulating hormone. Microinjection of the protein kinase C inhibitor protein caused a transient cessation of communication that spontaneously returned within 15-20 min. Treatment of cells with activators of protein kinase C, TPA or OAG for 60 min caused a significant reduction in communication that could be restored within 2-5 min by the subsequent injection of either the protein kinase C inhibitor or the protein kinase A catalytic subunit. With a longer exposure to either protein kinase C activator communication could not be restored and this appeared to be related to the absence of aggregates of connexin43 in membrane as detected immunologically. In cells injected with alkaline phosphatase communication stopped but returned either spontaneously within 20 min or within 2-3 min of injecting the cell with either the protein kinase A catalytic subunit or with protein kinase C. When untreated cells were injected with protein kinase C communication diminished or ceased within 5 min. Collectively these results demonstrate that cell-cell communication is regulated by both protein kinase A and C, but in a complex interrelated manner, quite likely by multiple phosphorylation of proteins within or regulating connexin-43 containing gap junctions.

Connexin-43-type gap junctions mediate communication between bone marrow stromal cells.

Several morphologic studies have suggested that gap junctions exist between bone marrow stromal cells. This possibility was examined by analysis of stromal cells present in the adherent layer of primary long-term lymphoid bone marrow cultures and in additional studies using a stromal cell line. Results showing that the fluorescent dye lucifer yellow, when microinjected into a single stromal cell, transferred between most other contacting stroma and that stromal cells were electronically coupled provided support that cell-cell communication occurs between these microenvironmental elements. Additional studies showed that transcripts for connexin (Cx) 43, but not for Cx26 or Cx32, were present in a stromal cell line. To examine the potential for regulated cell-cell communication between the stroma, cells were treated with interleukin-1 (IL-1), a cytokine known to affect stromal cell function, and the effects on dye transfer were examined. IL-1 treatment resulted in a reversible decrease in the ability of dye to transfer between stromal cells in contact. Taken together, these studies show that gap junctions exist between stromal cells and that their permeability can be regulated. However, gap junction-mediated cell-cell communication could not be shown between the stroma and developing lymphoid cells.

Fluorescence resonance energy transfer within a heterochromatic cAMP-dependent protein kinase holoenzyme under equilibrium conditions: new insights into the conformational changes that result in cAMP-dependent activation.

Previous studies of the ligand regulation of the cAMP-dependent protein kinase have demonstrated the cAMP-mediated dissociation of the holoenzyme by using nonequilibrium techniques; i.e., gel filtration, ion-exchange chromatography, and differential centrifugation. While physically mild, these could have caused weakly associated species to dissociate, thereby providing a potentially flawed interpretation of the mechanism of activation of the protein kinase. To assess this, the activation of the cAMP-dependent protein kinase has been monitored under equilibrium conditions using dipolar fluorescence energy transfer to measure changes in the proximity relations between the catalytic (C) and regulatory (R) subunits that compose the holoenzyme. Specifically, we prepared a heterochromatically labeled protein kinase type II holoenzyme, with the regulatory and catalytic subunits labeled with sulforhodamine and carboxyfluorescein, respectively, and monitored the exchange of electronic excitation energy between the C and R subunits by both donor lifetime and steady-state fluorescence. Biochemically, the heterochromatic holoenzyme was closely identical to the native protein with regard to cAMP-induced increase in catalytic activity, reassociation of C and R subunits, inhibition of catalytic activity by the specific protein kinase inhibitor (PKI), and observed dissociation examined by gel filtration upon cAMP addition. However, under equilibrium conditions, the energy-transfer measurements revealed that the addition of cAMP to this heterochromatic reporter complex promoted an estimated 10-A increase in the distance between the derivatization sites on C and R but not a dissociation of these subunits. Addition of PKI plus cAMP promoted full dissociation of the two subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

The hormone-induced regulation of contact-dependent cell-cell communication by phosphorylation.

Although there is insufficient evidence to propose an elaborate paradigm for the regulation of connexon gating, a simple model emerges from results of studies done to date. Basically, this centers around the most consistent findings: namely, that activation of pkA has an enhancing effect on cell communication while activation of pkC decreases that process. This fits well the reported phenomena associated with gap junctions, particularly those involving growth control. For example growth factors, including tumor promoters which work via pkC, usually reduce cell-cell communication whereas agents that decrease growth often raise cellular cAMP levels, which can lead to increased communication. It can be argued that this model is too simple because it fails to take into account other intracellular agents that are thought to alter junctional gating: cytoplasmic acidification, cellular free Ca2+, tyrosine protein kinases, and tentatively, pkG. Proton and Ca2+ transporting systems are mainly activated by serine/threonine protein kinases such as pkA and pkC. Some ion channels are not regulated by phosphorylation but instead are modulated by other ions. However, at the moment there is no evidence as to which ion-specific channels mediate the changes in cellular pH or Ca2+ that cause a loss in communication. Neither is it known whether pH or Ca2+ levels are in vivo regulators of the junctions. This is especially so as fairly high levels of injected Ca2+ pass through the gap junctions of viable cells. The role of tyrosine protein kinases in connexon gating may involve interaction with the pkA and pkC regulatory cascades. For example, the pkA inhibitor protein (pkI) is 80-90% inactivated when tyrosine-phosphorylated by the EGF receptor or pp50v-src (D. Walsh, personal communication). In this situation, activity of the C subunit of pkA could be enhanced, or the lifetime of its catalytic activity extended. In some systems, pp60v-src is known to activate the pkC pathway. Thus, tyrosine protein kinases may invoke pkA and pkC pathways; however, the amplitude of enzyme activation and the temporal kinetics of this process are unknown. The fact that gap junctions are regulated at the transcription level and probably at the protein level by protein kinases is of major interest. This is especially so as the only known molecular mechanism that gap junctional communication mediates is the activation of cAMP-dependent protein kinases by hormone-induced signals passed from receptor-bearing cells to receptorless partners.

Resistance to the cytolytic action of lymphotoxin and tumor necrosis factor coincides with the presence of gap junctions uniting target cells.

The resistance of target cells to the cytolytic action of lymphotoxin (LT) and recombinant tumor necrosis factor (rTNF) has been investigated by using clonally derived cell lines with defined gap junction-mediated, intercellular communication properties. Gap junction-competent Chinese hamster ovary cells are normally insensitive to the action of LT/TNF. However, treatment with 12-o-tetradecanoylphorbol-13-acetate, which promotes the loss of gap junctions, or culturing at low cell density to reduce intercellular contacts, significantly increased their sensitivity to LT/TNF. The LT/TNF-sensitive murine CL-1D and L929 cell lines, which in normal culture conditions are unable to form gap junctions, were not changed in their susceptibility to LT/TNF after treatment with phorbol ester or low culture density. However, the formation of gap junctions by CL-1D can be promoted by treatment with 8-bromo-cyclic adenosine monophosphate (1 mM), and this treatment completely suppressed the ability of LT and rTNF to kill CL-1D. Additionally, the LA25-normal rat kidney cell line, which is infected with a temperature-sensitive mutant of Rous sarcoma virus (LA25), is gap junction-competent and resistant to the effects of LT at the restrictive temperature (39 degrees C). However, when shifted to the permissive temperature (33 degrees C), LA25-normal rat kidney cells express the pp60v-src viral gene product, lose their ability to form gap junctions, and become sensitive to the lytic activity of LT. The results demonstrate that the expression of the retroviral pp60v-src, a tyrosine protein kinase, is sufficient to render cells susceptible to the lytic effects of LT and rTNF. Collectively, these experiments demonstrate a strong correlation between the resistance of target cells to the action of LT/TNF and their ability to cooperate metabolically through gap junctions. The results do not completely exclude the possibility that other mechanisms, such as LT receptor modulation, are also occurring under these experimental conditions. These data also suggest that a possible physiologic function of the stable cytotoxic lymphokines is to induce cytolysis/cytostasis of cells that have lost gap junctional contact, such as those in the process of mitosis or metastasis that have separated from the main tissue mass.

Cyclic AMP-dependent protein kinase-mediated desensitisation of adrenal tumour cells.

Y-1 mouse adrenal cortical tumour cells increase their production of steroids and cAMP while decreasing cell population growth in response to treatment with ACTH. With a fluorescent conjugate of the heat-stable protein kinase inhibitor, the time- and dose-dependent dissociation of cAMP-dependent protein kinase can be demonstrated following ACTH stimulation of Y-1 adrenal tumour cells, and the kinetics of its free catalytic subunits can be followed. After 60 min ACTH (6 X 10(-10) M) stimulation, a 4-fold rise in catalytic subunits was detected in the nucleus while a 2-fold increase was noted in the cytoplasm. When Y-1 cells had been previously treated with ACTH, they no longer secreted steroids to the same level in response to a subsequent exposure to ACTH. In addition to the altered steroidogenic response the cells become resistant to the effect of subsequent ACTH treatment on cell division and cAMP production as measured by protein kinase dissociation. Y-1 adrenal cells, that had been pretreated with ACTH, had an altered activation of protein kinase. Although there was an increase in cytoplasmic dissociation following subsequent ACTH stimulation of the pretreated cell, this increase was negligible when compared to that in the non-pretreated cultures. The nucleus of the ACTH-pretreated cell failed to significantly dissociate protein kinase following subsequent ACTH treatment. The data suggest that the phenomenon of desensitisation may be due to a decrease in dissociation of cAMP-dependent protein kinase, especially in the nucleus.

The inhibitor protein of the cAMP-dependent protein kinase-catalytic subunit interaction. Parameters of complex formation.

The formation of a complex between the catalytic subunit of the cAMP-dependent protein kinase and the Inhibitor Protein of this enzyme has been examined by means of nondenaturing gel electrophoresis. Two forms of complex were identified, both containing a 1:1 molar ratio of the component proteins. The formation of the major of the two forms is markedly enhanced by the presence of nucleotide triphosphate and divalent cation. Either Mg2+ or Mn2+ serves to promote complex formation. With Mg2+, only ATP is effective for enhancing complex formation, whereas with Mn2+ complex formation occurs to an equal extent with ATP, GTP, ITP, and adenyl-5'-yl imidodiphosphate. The formation of the two complexes is only minimally dependent upon nucleotide triphosphate. It is suggested that the two types of complex are a result of different species of catalytic subunit. Two principal forms of the complex have been detected occurring maximally in approximately a 2.5:1 ratio. In the accompanying paper (Fletcher, W.H., Van Patten, S.M., Cheng, H-C., and Walsh, D.A. (1986) J. Biol. Chem. 261, 5504-5513), we have described the use of a fluoresceinated derivative of catalytic subunit as a cytochemical probe to localize the Inhibitor Protein and the regulatory subunit of the protein kinase. The integrity of this fluorophore has been further characterized using the method of examining catalytic subunit-Inhibitor Protein interaction delineated here.

Cytochemical identification of the regulatory subunit of the cAMP-dependent protein kinase by use of fluorescently labeled catalytic subunit. Examination of protein kinase dissociation in hepatoma cells responding to 8-Br-cAMP stimulation.

Homogeneous catalytic subunit from the cAMP-dependent protein kinase, when derivatized with a fluorophore, was used as a cytochemical probe to locate intracellular sites of the protein kinase regulatory subunit. After conjugation, the fluoresceinated catalytic subunit (F:C), derivatized to a stoichiometry of approximately 1 mol/mol, retained near full activity as judged by specific activity and by titration against either regulatory subunit or Inhibitor Protein of the protein kinase. With this molecular probe the dissociated regulatory subunit was localized by direct cytochemistry in Reuber H-35 hepatoma cells that had been exposed, while intact, for 0-120 min to 10(-4) M 8-Br-cAMP. After stimulation, cultures were fixed and washed and then incubated for 16 h with F:C. Following 8-Br-cAMP stimulation, extensive binding of the probe to both cytoplasmic and nucleolar sites was observed. This binding was diminished but not eliminated when 50 microM cAMP was present during the incubation of the fixed cells with F:C that was eliminated by a 40-fold molar excess of underivatized catalytic subunit but not by heat-denatured catalytic subunit, and was not reduced by a 20-fold molar excess of cGMP-dependent protein kinase, examined plus or minus cGMP. Collectively, the results allow the conclusion that the F:C probe binds free regulatory subunit. The time course of its change with 8-Br-cAMP (measured as the difference between binding in the presence or absence of cAMP during the postfixation treatment) mirrors that previously reported for changes in the catalytic subunit in these cells, also identified cytochemically (Byus, C. V., and Fletcher, W.H. (1982) J. Cell Biol. 93, 727-734). The binding of the F:C probe, detected when cAMP is present during postfixation treatment, may possibly represent binding to free Inhibitor Protein of the cAMP-dependent protein kinase. If so, it was at a level of approximately 20% of the maximal level of detectable regulatory subunit, and it also showed cytosolic and nucleolar localization.

Receptor mediated action without receptor occupancy.

The relationship between occupancy of a specific cell surface receptor and actions initiated in the same cell by that event was tested on ovarian granulosa cells exposed to hCG. Hormone bound to its receptor was identified by immunocytochemistry or by autoradiography and the dissociation of cAMP-dependent protein kinase was followed by direct cytochemistry. Only 25-30% of the granulosa cells bound hCG and in each instance this resulted in protein kinase dissociation. Cells that did not bind hCG nevertheless dissociated protein kinase if they contacted a cell that had bound hormone and dissociated enzyme. Cells that neither bound hormone, nor contacted cells that did so, failed to dissociate protein kinase. These observations establish, in individual cells, a direct relationship of receptor occupancy to receptor-mediated action and indicate that this event can be communicated to receptorless cells, presumably by gap junctions, thereby amplifying the response to hormone. Similar processes may occur in other tissues wherein receptor-bearing cells are capable of intercellular communication.

Morphologic analysis of long-term bone marrow cultures that support B-lymphopoiesis or myelopoiesis.

A comparative morphological analysis of the Whitlock-Witte long-term B-cell culture and the predominantly myeloid Dexter long-term bone marrow culture demonstrates that similarities and differences exist between the two systems. Cells from the long-term B-cell cultures have a characteristic lymphoid morphology, whereas those from the Dexter cultures are predominantly granulocytes and macrophages along with a few undifferentiated blast cells. A multilayered stromal cell layer is a common feature of both systems. Scanning electron micrographs show the cells in this layer to be large, irregularly shaped and flattened. The data further indicate that there are unique features in the relationship between developing B cells and stromal cells in the long-term B-cell cultures. Large, mononuclear cells are present that have numerous membrane infoldings within which numerous lymphoid cells lie. The relationship of these cells to macrophages and epithelial/reticular cells is discussed.