Development of cGAMP-Luc, a sensitive and precise coupled enzyme assay to measure cGAMP in complex biological samples.

2',5'/3',5'-cGMP-AMP (cGAMP) is a second messenger produced in response to cytosolic dsDNA that activates the stimulator of interferon genes (STING) pathway. We recently discovered that cGAMP is exported by cancer cells and that this extracellular signal is an immunotransmitter key to tumor detection and elimination by the innate immune system. The enhancement of extracellular cGAMP levels therefore holds great promise for managing cancer. However, there is still much more to understand about the basic biology of cGAMP before its full therapeutic potential can be realized. To answer these questions, we must be able to detect and quantitate cGAMP with an assay that is high-throughput, sensitive, and precise. Existing assays fall short of these needs. Here, we describe the development of cGAMP-Luc, a coupled enzyme assay that relies on the degradation of cGAMP to AMP by ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) and an optimized assay for the detection of AMP by luciferase. We also developed STING-CAP, a STING-mediated method to concentrate and purify cGAMP from any type of biological sample. We conclude that cGAMP-Luc is an economical high-throughput assay that matches the accuracy of and surpasses the detection limit of MS, the current gold standard of cGAMP quantitation. We propose that cGAMP-Luc is a powerful tool that may enable discoveries that advance insights into extracellular cGAMP levels in healthy and diseased tissues, such as cancer.

Mass spectrometric characterization of cyclic dinucleotides (CDNs) in vivo.

Cyclic dinucleotides (CDNs) are key secondary messenger molecules produced by cyclic dinucleotide synthases that trigger various cellular signaling cascades from bacteria to vertebrates. In mammals, cyclic GMP-AMP synthase (cGAS) has been shown to bind to intracellular DNA and catalyze the production of the dinucleotide 2'3' cGAMP, which signals downstream effectors to regulate immune function, interferon signaling, and the antiviral response. Despite the importance of CDNs, sensitive and accurate methods to measure their levels in vivo are lacking. Here, we report a novel LC-MS/MS method to quantify CDNs in vivo. We characterized the mass spectrometric behavior of four different biologically relevant CDNs (c-di-AMP, c-di-GMP, 3'3' cGAMP, 2'3' cGAMP) and provided a means of visually representing fragmentation resulting from collision-induced dissociation at different energies using collision energy breakdown graphs. We then validated the method and quantified CDNs in two in vivo systems, the bacteria Escherichia coli OP50 and the killifish Nothobranchius furzeri. We found that optimization of LC-MS/MS parameters is crucial to sensitivity and accuracy. These technical advances should help illuminate physiological and pathological roles of these CDNs in in vivo settings. Graphical abstract.

The Role of the Anion in Salt (NaCl) Detection by Mouse Taste Buds.

How taste buds detect NaCl remains poorly understood. Among other problems, applying taste-relevant concentrations of NaCl (50-500 mm) onto isolated taste buds or cells exposes them to unphysiological (hypo/hypertonic) conditions. To overcome these limitations, we used the anterior tongue of male and female mice to implement a slice preparation in which fungiform taste buds are in a relatively intact tissue environment and stimuli are limited to the taste pore. Taste-evoked responses were monitored using confocal Ca2+ imaging via GCaMP3 expressed in Type 2 and Type 3 taste bud cells. NaCl evoked intracellular mobilization of Ca2+ in the apical tips of a subset of taste cells. The concentration dependence and rapid adaptation of NaCl-evoked cellular responses closely resembled behavioral and afferent nerve responses to NaCl. Importantly, taste cell responses were not inhibited by the diuretic, amiloride. Post hoc immunostaining revealed that >80% of NaCl-responsive taste bud cells were of Type 2. Many NaCl-responsive cells were also sensitive to stimuli that activate Type 2 cells but never to stimuli for Type 3 cells. Ion substitutions revealed that amiloride-insensitive NaCl responses depended on Cl- rather than Na+ Moreover, choline chloride, an established salt taste enhancer, was equally effective a stimulus as sodium chloride. Although the apical transducer for Cl- remains unknown, blocking known chloride channels and cotransporters had little effect on NaCl responses. Together, our data suggest that chloride, an essential nutrient, is a key determinant of taste transduction for amiloride-insensitive salt taste.SIGNIFICANCE STATEMENT Sodium and chloride are essential nutrients and must be regularly consumed to replace excreted NaCl. Thus, understanding salt taste, which informs salt appetite, is important from a fundamental sensory perspective and forms the basis for interventions to replace/reduce excess Na+ consumption. This study examines responses to NaCl in a semi-intact preparation of mouse taste buds. We identify taste cells that respond to NaCl in the presence of amiloride, which is significant because much of human salt taste also is amiloride-insensitive. Further, we demonstrate that Cl-, not Na+, generates these amiloride-insensitive salt taste responses. Intriguingly, choline chloride, a commercial salt taste enhancer, is also a highly effective stimulus for these cells.

Mass Spectrometric Analysis of Non-canonical Cyclic Nucleotides.

Contemporary investigations regarding the (patho)physiological roles of the non-canonical cyclic nucleoside monophosphates (cNMP) cytidine 3',5'-cyclic monophosphate (cCMP) and uridine 3',5'-cyclic monophosphate (cUMP) have been hampered by the lack of highly specific and sensitive analytic methods for these analytes. In addition, the existence of 2',3'-cNMP besides 3',5'-cNMP has been described recently. HPLC coupled with tandem mass spectrometry (HPLC-MS/MS) is the method of choice for identification and quantification of low-molecular weight endogenous metabolites. In this chapter, recommendations for an HPLC-MS/MS method for 3',5'- and 2',3'-cNMP are summarized.

Chemical shift assignments of calmodulin bound to the ß-subunit of a retinal cyclic nucleotide-gated channel (CNGB1).

Rod cyclic nucleotide-gated (CNG) channels are formed by two protein subunits (CNGA1 and CNGB1). Calmodulin (CaM) binds to the cytosolic regulatory domain of CNGB1 and decreases the open probability of CNGA1/CNGB1 channels. The CaM binding site within bovine CNGB1 (residues 679-702) binds tightly to Ca2+-bound CaM, which promotes Ca2+-induced inactivation of CNGA1/CNGB1 channels in retinal rods. We report complete NMR chemical shift assignments of Ca2+-saturated CaM bound to the CaM-binding domain of CNGB1 (BMRB no. 51222).

Chemical shift assignments of calmodulin bound to a C-terminal site (residues 1120-1147) in the ß-subunit of a retinal cyclic nucleotide-gated channel (CNGB1).

Retinal cyclic nucleotide-gated (CNG) channels consist of two protein subunits (CNGA1 and CNGB1). Calmodulin (CaM) binds to two separate sites within the cytosolic region of CNGB1: CaM binding to an N-terminal site (human CNGB1 residues 565-587, called CaM1) decreases the open probability of CNG channels at elevated Ca2+ levels in dark-adapted photoreceptors, whereas CaM binding to a separate C-terminal site (CNGB1 residues 1120-1147, called CaM2) may increase channel open probability in light activated photoreceptors. We recently reported NMR chemical shift assignments of Ca2+-saturated CaM bound to the CaM1 site of CNGB1 (BMRB no. 51222). Here, we report complete NMR chemical shift assignments of Ca2+-saturated CaM bound to the C-terminal CaM2 site of CNGB1 (BMRB no. 51447).

The influence of intracellular levels of cyclic nucleotides on cell proliferation and the induction of antibody synthesis.

The intracellular ratio of adenosine 3',5'-cyclic monophosphate (cyclic AMP) to guanosine 3',5'-cyclic monophosphate (cyclic GMP) may control the developmental pathway followed by antibody-forming cell (AFC) precursors. The evidence for this is derived from several different types of experiments. First lipopolysaccharide (LPS) which is mitogenic for B lymphocytes, stimulates rapid, transient changes in intracellular levels of cyclic GMP but not cyclic AMP when added to mouse spleen cultures. Cyclic GMP itself stimulates DNA synthesis in these cultures, suggesting that the intracellular changes in cyclic GMP levels are involved in the mitogenic signal delivered by LPS to cells. The absolute amounts of cyclic nucleotides may vary widely in different cells under various conditions, however, the intracellular ratio of cyclic AMP to cyclic GMP is always high in nondividing cells and low in dividing cells. AFC precursors appear to respond to antigen in the absence of T-cell activity by inactivation (1-7). In the response to antigen in the presence of specific T cells, precursor cells proliferate and mature to AFC. Raising intracellular levels of cyclic AMP inhibits cell proliferation and leads to precursor cell inactivation (14, 15). It is suggested that the interaction of antigen with immunoglobulin receptors on the surface of precursors cells leads to the stimulation of adenylate cyclase activity and initiates the inactivation pathway. Since cyclic GMP stimulates immune responses in T-cell-depleted cultures (14, 15) and increasing cyclic GMP levels appear to be involved in the delivery of a mitogenic signal to cells, it is suggested that T-helper cells deliver a signal to precursor cells via the stimulation of guanylate cyclase to initiate the inductive pathway. It is suggested that it is the intracellular ratio of cyclic AMP to cyclic GMP that regulates the fate of precursor cells, not the absolute level of one cyclic nucleotide.

A STING-based biosensor affords broad cyclic dinucleotide detection within single living eukaryotic cells.

Cyclic dinucleotides (CDNs) are second messengers conserved across all three domains of life. Within eukaryotes they mediate protective roles in innate immunity against malignant, viral, and bacterial disease, and exert pathological effects in autoimmune disorders. Despite their ubiquitous role in diverse biological contexts, CDN detection methods are limited. Here, using structure guided design of the murine STING CDN binding domain, we engineer a Förster resonance energy transfer (FRET) based biosensor deemed BioSTING. Recombinant BioSTING affords real-time detection of CDN synthase activity and inhibition. Expression of BioSTING in live human cells allows quantification of localized bacterial and eukaryotic CDN levels in single cells with low nanomolar sensitivity. These findings establish BioSTING as a powerful kinetic in vitro platform amenable to high throughput screens and as a broadly applicable cellular tool to interrogate the temporal and spatial dynamics of CDN signaling in a variety of infectious, malignant, and autoimmune contexts.

Thiophenecarboxylate suppressor of cyclic nucleotides discovered in a small-molecule screen blocks toxin-induced intestinal fluid secretion.

We carried out a "pathway" screen of 50,000 small molecules to identify novel modulators of cAMP signaling. One class of compounds, the 2-(acylamino)-3-thiophenecarboxylates, strongly suppressed cAMP and cGMP in multiple cell lines in response to different agonists acting on G-protein-coupled receptors, adenylyl cyclase, and guanylyl cyclase. The best compounds from structure-activity analysis of 124 analogs, including several synthesized chiral analogs, had and IC(50) of <5 microM for suppression of agonist-induced cAMP and cGMP elevation. Measurements of cAMP, cGMP, and downstream signaling in response to various activators/inhibitors suggested that the 2-(acylamino)-3-thiophenecarboxylates function as nonselective phosphodiesterase activators, although it was not determined whether their action on phosphodiesterases is direct or indirect. The 2-(acylamino)-3-thiophenecarboxylates suppressed CFTR-mediated Cl(-) current in T84 colonic cells in response to cholera and Escherichia coli (STa) toxins, and prevented intestinal fluid accumulation in a closed-loop mouse model of secretory diarrhea. They also prevented cyst growth in an in vitro renal epithelial cell model of polycystic kidney disease. The 2-(acylamino)-3-thiophenecarboxylates represent the first small-molecule cyclic nucleotide suppressors, whose potential therapeutic indications include secretory diarrheas, polycystic kidney disease, and growth inhibition of cAMP-dependent tumors.

Cyclic nucleotide-regulated proliferation and differentiation vary in human hematopoietic progenitor cells derived from healthy persons, tumor patients, and chronic myelocytic leukemia patients.

Although circulating hematopoietic progenitor cells (HPCs) are frequently used in therapeutic approaches, many aspects of their cellular biochemistry are still unclear. In the present study, the effects of cyclic nucleotide-elevating agents on HPC proliferation and differentiation were investigated. HPCs from different sources, including healthy persons, patients with tumors (medulloblastoma, seminoma, or multiple myeloma), and patients with chronic myelocytic leukemia (CML), were compared. HPCs were isolated by standard leukapheresis procedures and analyzed for proliferation and differentiation into the megakaryocytic and granulocytic lineages. HPCs contained high concentrations of cyclic guanosine monophosphate (cGMP)-dependent and cyclic adenosine monophosphate (cAMP)-dependent protein kinases G and A (PKG and PKA, respectively). Whereas PKG was partly down-regulated during culture, the PKA level remained constant. Stimulation of PKG in HPCs isolated from healthy donors or tumor patients resulted in a biphasic reaction: low cGMP concentrations inhibited proliferation and stimulated differentiation into megakaryocytes, whereas high concentrations revealed the opposite effect. In contrast, differentiation into granulocytes was inhibited in a concentration-dependent manner. Stimulation of PKA inhibited HPC differentiation; however, HPC proliferation was inhibited in controls and stimulated in HPCs from tumor patients. HPCs isolated from CML patients showed a nonhomogeneous reaction pattern to both cyclic nucleotides with high variability between the individual donors. We demonstrated the importance of the source of HPCs for the investigation of proliferation and differentiation. Cyclic nucleotide-regulated pathways are clearly involved in HPC proliferation and differentiation. Pharmacological strategies using cyclic nucleotide-elevating substances to influence HPC growth and differentiation in the bone marrow might support current strategies in HPC recovery from the peripheral blood.

Expression of crustacean (Callinectes sapidus) molt-inhibiting hormone in Escherichia coli: characterization of the recombinant peptide and assessment of its effects on cellular signaling pathways in Y-organs.

A neuropeptide, molt-inhibiting hormone (MIH), negatively regulates the synthesis of ecdysteroid molting hormones by crustacean Y-organs. We report here the expression of blue crab (Callinectes sapidus) MIH in Escherichia coli. Bacteria were transformed with an expression plasmid containing a cDNA insert encoding MIH. After induction of protein synthesis, recombinant MIH (recMIH) was detected in the insoluble fraction of cell lysates. The insoluble recMIH was refolded and purified by reversed-phase high performance liquid chromatography (RP-HPLC). The refolded peptide was MIH-immunoreactive and comigrated with native MIH on RP-HPLC. Mass and CD spectral analyses showed the mass number and secondary structure of the recombinant peptide were as predicted for MIH. Bioassays showed recMIH dose-dependently suppresses ecdysteroid synthesis by Y-organs. The combined results suggest that recMIH is properly folded. In subsequent experiments, recMIH was used to assess cellular signaling pathways linked to MIH-mediated suppression of ecdysteroidogenesis. Incubation of Y-organs with recMIH produced an increase in intracellular cGMP content, but had no effect on intracellular cAMP. Further, a cGMP analog significantly suppressed ecdysteroid production, but neither cAMP analogs nor an activator of adenylyl cyclase had a detectable effect on ecdysteroidogenesis. The results are consistent with the hypothesis that MIH-induced suppression of ecdysteroidogenesis in Y-organs of C. sapidus is mediated by a cGMP second messenger. We anticipate recMIH will be a useful tool for additional studies of the cellular actions and physiological functions of MIH.

Relationship between the levels of cyclic cytidine 3':5'-monophosphate, cyclic guanosine 3':5'-monophosphate, and cyclic adenosine 3':5'-monophosphate in urines and leukocytes and the type of human leukemias.

Cyclic cytidine 3':5'-monophosphate (cyclic CMP), cyclic guanosine 3':5'-monophosphate (cyclic GMP), and cyclic adenosine 3':5'-monophosphate (cyclic AMP) contents of leukocytes and urines of leukemic patients have been investigated. We have studied four types of leukemia: acute myeloblastic leukemia; chronic myelocytic leukemia; acute lymphoblastic leukemia; and chronic lymphocytic leukemia. As controls, the cyclic nucleotide content of leukocytes and urines of healthy volunteers and patients with solid tumors selected for their normal hemogram has been determined. It has also been measured in phytohemagglutinin-stimulated lymphocytes. Our data show that: (a) the concentration of cyclic CMP is always lower than that of cyclic GMP or cyclic AMP; (b) in urines, the concentrations of the three nucleotides are higher in patients than in healthy volunteers, the greatest differences being observed between the cyclic CMP concentrations of acute leukemia patients and controls; and (c) in white blood cells, cyclic AMP concentration is lower in leukemic than in normal cells. The cyclic GMP concentration is the same everywhere except in monoblastic cells and leukocytes from solid tumor patients. High cyclic CMP levels are associated only with acute leukemia, whether myeloblastic, monoblastic, or lymphoblastic, a fact which suggests that cyclic CMP could be a biochemical marker of hematopoietic stem cell malignancy.

Requirements for and kinetics of growth arrest of neoplastic cells by confluent 10T1/2 fibroblasts induced by a specific inhibitor of cyclic adenosine 3':5'-phosphodiesterase.

This study was conducted to further characterize the previously described phenomenon of growth inhibition of neoplastically transformed C3H/10T1/2 cells (T10T1/2) by nontransformed C3H/10T1/2 clone 8 mouse embryo fibroblast (10T1/2) cells in the presence of inhibitors of cyclic adenosine 3':5'-monophosphate (cAMP) phosphodiesterase. The cAMP phosphodiesterase inhibitor dl-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (RO20-1724) was shown to be completely nontoxic to T10T1/2 cells at 10(-4) M, yet when added to mixed cultures of T10T1/2 cells and postconfluence growth-arrested 10T1/2 cells, colony formation and [3H]thymidine incorporation into T10T1/2 cells were virtually eliminated. This effect was dose dependent and was reversible upon drug withdrawal. In 10T1/2 cells, RO20-1724 caused a dose-dependent increase in cAMP levels from about 5 to 150 pmol/10(6) cells; in T10T1/2 cells, 10(-4) M drug treatment caused a 5-fold elevation in cAMP without a clear dose dependency. Cyclic guanosine 3':5'-monophosphate levels in 10T1/2 cells fell by 50% with drug treatment but were unmeasurable in T10T1/2 cells. When intracellular cyclic AMP levels were elevated by the adenyl cyclase stimulator forskolin, growth inhibition of T10T1/2 cells was again induced in mixed cultures but was not observed when added to T10T1/2 cells alone. Addition of RO20-1724 to low concentrations of forskolin produced a greater than additive effect on growth inhibition. Growth inhibition of T10T1/2 cells by RO20-1724 was shown to (a) require contact with, or extremely close proximity to, a confluent monolayer of 10T1/2 cells, (b) be maximum when seeded upon a growth-inhibited monolayer and not an actively growing 10T1/2 culture, and (c) not be decreased by continuous agitation of the culture medium, indicating that readily diffusible inhibitory factors are not involved. A model is presented whereby transformed cells can respond to but cannot themselves generate growth-inhibitory signals produced by post-confluence growth-inhibited nontransformed cells. The existence of these cellular interactions may well explain problems in the quantitation of transformed foci encountered in the use of this cell line as an assay system for chemical and physical carcinogens.

Patch cramming reveals the mechanism of long-term suppression of cyclic nucleotides in intact neurons.

To understand cyclic nucleotide dynamics in intact cells, we used the patch-cramming method with cyclic nucleotide-gated channels as real-time biosensors for cGMP. In neuroblastoma and sympathetic neurons, both muscarinic agonists and nitric oxide (NO) rapidly elevate cGMP. However, muscarinic agonists also elicit a long-term (2 hr) suppression (LTS) of subsequent cGMP responses. Muscarinic agonists elevate cGMP by triggering Ca2+ mobilization, which activates NO synthase to produce NO, leading to the activation of soluble guanylate cyclase (sGC). Here we examine the mechanism of LTS. Experiments using direct intracellular cGMP injection demonstrate that enhancement of phosphodiesterase (PDE) activity, rather than depression of sGC activity, is responsible for LTS. Biochemical measurements show that both cGMP and cAMP content is suppressed, consistent with the involvement of a nonselective PDE. Application of pharmacological agents that alter Ca2+ mobilization from intracellular stores and experiments involving injection of the Ca2+ chelator BAPTA show that Ca2+ mobilization is necessary and sufficient for LTS induction but also show that LTS maintenance is Ca2+-independent. Protein phosphatase injection reverses LTS, and specific inhibitors of Ca2+/calmodulin kinase II (CaMKII) prevent induction and inhibit maintenance. The switch between the Ca2+ dependence of LTS induction to the Ca2+ independence of LTS maintenance is consistent with CaMKII autophosphorylation, similar to proposed mechanisms of hippocampal long-term potentiation. Because the molecular machinery underlying LTS is common to many cells, LTS may be a widespread mechanism for long-term silencing of cyclic nucleotide signaling.

Prevention of diabetes-induced microangiopathy by human tissue kallikrein gene transfer.

BACKGROUND: Microvascular insufficiency represents a major cause of end-organ failure among diabetics. METHODS AND RESULTS: In streptozotocin-induced diabetic mice, we evaluated the potential of human tissue kallikrein (hTK) gene as a sole therapy against peripheral microangiopathy. Local delivery of hTK gene halted the progression of microvascular rarefaction in hindlimb skeletal muscle by inhibiting apoptosis, thus ensuring an improved hemodynamic recovery in case of supervening vascular occlusion. The curative action of hTK did not necessitate insulin supplementation. Application of gene therapy at a stage of established microangiopathy stimulated vascular regeneration. CONCLUSIONS: Our studies indicate that hTK may represent a useful tool for the treatment of microvascular complications in diabetics.

[A metabolic function of the lungs in postoperative changes of vascular regulation in patients with occlusive lesions of the abdominal aorta and major arteries of the lower limbs].

AIM: To study a pathogenetic role of endogenous biologically active substances (EBAS) in the development of a vascular response and EBAS metabolism in the lungs of patients with surgical vascular pathology. MATERIAL AND METHODS: Content of serotonin, histamine, epinephrine, prostaglandins, cyclic nucleotides were studied in 84 patients with occlusive lesions of the abdominal aorta and major arteries before and after reconstructive surgery. RESULTS: The greatest changes in concentrations of EBAS and their metabolism in the lungs were observed in an immediate postoperative period in patients with baseline arterial hypertension and without it. These changes reflected initial disorders of normal relations of EBAS and influence of perioperative factors. Periods of aortal cross-clamping and blood recirculation in ischemic limbs were key factors of critical activation of the systems of humoral circulation regulation. CONCLUSION: Quantitative and qualitative changes in EBAS balance lead to enhancement of peripheral resistance and development of arterial hypertension.

From canonical to non-canonical cyclic nucleotides as second messengers: pharmacological implications.

This review summarizes our knowledge on the non-canonical cyclic nucleotides cCMP, cUMP, cIMP, cXMP and cTMP. We place the field into a historic context and discuss unresolved questions and future directions of research. We discuss the implications of non-canonical cyclic nucleotides for experimental and clinical pharmacology, focusing on bacterial infections, cardiovascular and neuropsychiatric disorders and reproduction medicine. The canonical cyclic purine nucleotides cAMP and cGMP fulfill the criteria of second messengers. (i) cAMP and cGMP are synthesized by specific generators, i.e. adenylyl and guanylyl cyclases, respectively. (ii) cAMP and cGMP activate specific effector proteins, e.g. protein kinases. (iii) cAMP and cGMP exert specific biological effects. (iv) The biological effects of cAMP and cGMP are terminated by phosphodiesterases and export. The effects of cAMP and cGMP are mimicked by (v) membrane-permeable cyclic nucleotide analogs and (vi) bacterial toxins. For decades, the existence and relevance of cCMP and cUMP have been controversial. Modern mass-spectrometric methods have unequivocally demonstrated the existence of cCMP and cUMP in mammalian cells. For both, cCMP and cUMP, the criteria for second messenger molecules are now fulfilled as well. There are specific patterns by which nucleotidyl cyclases generate cNMPs and how they are degraded and exported, resulting in unique cNMP signatures in biological systems. cNMP signaling systems, specifically at the level of soluble guanylyl cyclase, soluble adenylyl cyclase and ExoY from Pseudomonas aeruginosa are more promiscuous than previously appreciated. cUMP and cCMP are evolutionary new molecules, probably reflecting an adaption to signaling requirements in higher organisms.

Cyclic AMP, cyclic GMP, and glucocorticoids as potential metabolic regulators of epidermal proliferation and differentiation.

The two cyclic nucleotides, cyclic AMP and cyclic GMP, appear to be central to the metabolic regulation of cell proliferation and differentiation in various cells. Moreover, in many systems glucocorticoids appear to act in concert with or parallel to cyclic AMP. The available evidence suggests that these three molecular species--cyclic AMP, cyclic GMP, and glucocorticoids--may be essential to the normal regulation of epidermal proliferation and differentiation. In 1970, we suggested that perturbed epidermal homeostasis, exemplified by psoriasis, might be associated with low cellular levels of cyclic AMP and, in 1972, with high levels of cyclic GMP as well. Subsequent measurements of these two cyclic nucleotides in our laboratory showed a probable reduction in the cyclic AMP/cyclic GMP ratio in lesional psoriatic tissue. This led to the hypothesis that the cardinal features of psoriatic epidermis--glycogen accumulation, excessive proliferation, and reduced cell specialization--are the results of this reduced ratio. A corollary of this hypothesis was that a psoriatic lesion could not begin or exist without this altered cyclic nucleotide ratio. Recently, four different agents--lithium, a beta adrenergic blocking agent, antimalarials, and iodide--have been found to exacerbate psoriasis and to reduce the formation of cyclic AMP in various tissues. Consequently we believe that cyclic nucleotides are of central importance in the pathogenesis of the epidermal component of psoriasis.

New evidence for an acute role of protein kinase in hCG action.

The present experiments were designed to study whether exogenous hCG could elicit acute changes in the ovarian concentration of soluble cAMP-dependent protein kinases temporally related to binding of hCG and intracellular accumulation of cAMP. Cyclic AMP dependent protein kinase activity decreased five-fold within 5 to 30 min after intravenous administration of highly purified hCG to pseudopregnant rats. Moreover cAMP dependent protein kinase activity was totally suppressed with 0.5 IU hCG, whereas tissue concentration of cAMP continued to increase throughout the dose range (0.05-5.0 IU) of hCG used in the present studies. A marked fall in cAMP-dependent protein kinase activity had occurred before there was a significant change in intracellular accumulation of cAMP, possibly reflecting intracellular compartmentalization of cAMP. Inhibitors of protein did not affect the hCG-induced changes in tissue concentrations of cAMP and soluble cAMP dependent protein kinase activity but did suppress the recovery of cAMP dependent protein kinase activity to pretreatment levels. Cyclic AMP dependent protein kinases appear to play a significant role in mediating hormonal action in vivo. In addition the present studies suggest that, protein kinases may protect the cell from excessive hormonal stimulation.

Some cytological and initial biochemical observations on photoreceptors in retinas of rds mice.

As a control for biochemical studies in progress, an ultrastructural study has been carried out on the deteriorating, 21-day photoreceptors of the 020/Cpb strain of mice, homozygous for retinal degeneration, slow (rds). At 21 postnatal days, outer segments were essentially lacking, but cilia erupting from the inner segments were common. A low percentage of cilia bore small cytoplasmic masses containing a few layered membranes, and rare inner segments possessed spherical aggregations of multilayered membranes. Pigment epithelial cells also possessed membranous aggregations in presumed phagosomes. While other parts of photoreceptors possessed the usual organelles of normal rods, inner segments were reduced in volume, and the layer of photoreceptor synaptic terminals was thinner. Mutant 21-day retinas possessed about two-thirds of the protein of normal 21-day retinas but 50% more protein than "rodless" (rd/rd) 21-day retinas. Surprisingly, while dark-adapted rds retinas possessed markedly lower levels of cyclic GMP as compared to controls, light-adaptation significantly reduced cyclic GMP and cyclic AMP levels, and biochemical data point to persistent light-modulated cyclic nucleotide levels in the photoreceptors.