Metabolism of 1alpha,25-dihydroxyvitamin D(3) and its C-3 epimer 1alpha,25-dihydroxy-3-epi-vitamin D(3) in neonatal human keratinocytes.

We previously reported that 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is metabolized into 1alpha,25-dihydroxy-3-epi-vitamin D(3) [1alpha,25(OH)(2)-3-epi-D(3)] in primary cultures of neonatal human keratinocytes. We now report that 1alpha,25(OH)(2)-3-epi-D(3) itself is further metabolized in human keratinocytes into several polar metabolites. One of the polar metabolite was unequivocally identified as 1alpha,23,25-trihydroxy-3-epi-vitamin D(3) by mass spectrometry and its sensitivity to sodium periodate. Three of the polar metabolites were identified as 1alpha,24,25-trihydroxy-3-epi-vitamin D(3), 1alpha,25-dihydroxy-24-oxo-3-epi-vitamin D(3) and 1alpha,23,25-trihydroxy-24-oxo-3-epi-vitamin D(3) by comigration with authentic standards on both straight and reverse phase HPLC systems. In addition to the polar metabolites, 1alpha,25(OH)(2)-3-epi-D(3) was also metabolized into two less polar metabolites. A possible structure of either 1alphaOH-3-epi-D(3)-20,25-cyclic ether or 1alphaOH-3-epi-D(3)-24,25-epoxide was assigned to one of the less polar metabolites through mass spectrometry. Thus, we indicate for the first time that 1alpha,25(OH)(2)-3-epi-D(3) is metabolized in neonatal human keratinocytes not only via the same C-24 and C-23 oxidation pathways like its parent, 1alpha,25(OH)(2)D(3); but also is metabolized into a less polar metabolite via a pathway that is unique to 1alpha,25(OH)(2)-3-epi-D(3).

Modulation of epidermal differentiation, tissue inflammation, and T-lymphocyte infilitration in psoriatic plaques by topical calcitriol.

Psoriasis is characterized by immune activation, increased proliferation and abnormal differentiation of keratinocytes. The reported anti-psoriatic mechanisms of action in vivo of vitamin D analogues include reduction of keratinocyte proliferation and induction of keratinocyte terminal differentiation. We investigated whether the anti-psoriatic effect of the natural active vatamin D analogue, calcitriol, applied topically, is due to direct effects on keratinocytes alone or also due to immunoregulatory effects of calcitriol. Psoriasis patients were treated with topical calcitriol (0.005%) and a vehicle control for 8 weeks. Disease activity was assessed by a severity index and quantitative histopathological markers. In vitro studies of lymphocyte proliferation and gamma interferon secretion and of keratinocyte proliferation complemented the clinicohistopathologic studies. A heterogeneous response to calcitriol treatment could be segregated based upon elimination of K-16 keratin expression. Calcitriol treatment decreased keratinocyte proliferation, normalized keratinocyte differentiation and decreased immune activation in plaques. The histologic response to vitamin D treatment of psoriasis includes suppression of both immune and keratinocyte activation in situ. These studies provide a basis for rational combination of anti-psoriatic treatments and for the design of new vitamin D analogues to treat psoriasis.

Direct activation of protein kinase C by 1 alpha,25-dihydroxyvitamin D3.

The key metabolite of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 (1,25-D3), induces rapid cellular responses that constitute a so-called "non-genomic" response. This effect is distinguished from its "classic" genomic role in calcium homeostasis involving the nuclear 1,25-D3 receptor. Evidence is presented that protein kinase C (PKC) is directly activated by 1,25-D3 at physiological concentrations (EC50 = 16 +/- 1 nM). The effect was demonstrable with single PKC-alpha, -gamma, and -epsilon isoform preparations, assayed in a system containing only purified enzyme, substrate, co-factors, and lipid vesicles, from which it is inferred that a direct interaction with the enzyme is involved. The finding that calcium-independent isoform PKC-epsilon was also activated by 1,25-D3 shows that the calcium binding C2 domain is not required. The level of 1,25-D3-induced activation, paired with either diacylglycerol or 4 beta-12-O-tetradecanoylphorbol-13-acetate, was greater than that achievable by any individual activator alone, each at a saturating concentration, a result that implies two distinct activator sites on the PKC molecule. Phosphatidylethanolamine present in the lipid vesicles potentiated 4 beta-12-O-tetradecanoylphorbol-13-acetate- and diacylglycerol-induced PKC activities, whereas 1,25-D3-induced activity decreased, consistent with 1,25-D3-activated PKC possessing a distinct conformation. The results suggest that PKC is a "membrane-bound receptor" for 1,25-D3 and that it could be important in the control of non-genomic cellular responses to the hormone.

Effects of ORG 2766, a neurotrophic ACTH4-9 analogue, in neuroblastoma cells.

Treatment of Neuro2a cells with drugs known to affect the integrity of microfilaments and microtubules, as well as with a calcium ionophore produced damage to the cellular membrane that was quantifiable by measuring the release of LDH into the culture medium. Concurrent exposure of the cells to ORG 2766 was found to modulate the release of LDH in a dose- and time-dependent fashion. ORG 2766 treatment was also able to reduce the basal release of LDH into the culture medium. [table: see text] The ORG 2766-induced reduction in LDH release was not due to down-regulation of protein synthesis. The peptide produced significant increases in protein synthesis relative to control conditions at concentrations of 10(-11) to 10(-6) M with 10(-8) M being an optimal dose. SDS-PAGE and 2-D PAGE analysis showed that de novo synthesis of most polypeptides was increased by about 40%. Additionally, a family of polypeptides tentatively identified as actins appear to undergo ORG 2766-dependent post translational charge modifications. These data are consistent with the hypothesis that regulation of transcription and/or translation are mechanisms important to the neurotrophic actions of ORG 2766.

Signaling pathways for vitamin D-induced differentiation: implications for therapy of proliferative and neoplastic diseases.

The physiological form of vitamin D, 1 alpha,25-dihydroxyvitamin D3 (1,25D3) is not only a powerful regulator of calcium homeostasis but also a hormone with important roles in cell growth and differentiation. The differentiating effects of 1,25D3 have been studied extensively in a large number of in vitro systems but primarily using cultured leukemia cells, keratinocytes, and bone cells. In spite of this attention, the understanding of the mechanisms that lead to the diverse forms of cell differentiation by 1,25D3 is fragmentary. It seems likely, however, that the effects of 1,25D3 on calcium homeostasis and on cell differentiation are both signaled by direct effects on the cell membrane and by the transduction of the signal to the genome. The direct effect may be mediated by a hypothetical membrane recognition element, the G proteins, phosphoinositide metabolism, and protein kinase C (PKC). Signals to the nucleus may include PKC, the Ca2+ gradient, and protein phosphorylation cascades, but the protein receptor (VDR) plays a principal role in this form of signal transduction. The VDR interacts with the promoter elements of a number of nuclear genes, either as a homodimer or a heterodimer with a retinoid or orphan receptors. The key nuclear responses include the increased expression of phenotype-specifying proteins and the appearance of G1/S and G2+M cell cycle blocks, which result in cessation of cell proliferation. The attempts to improve control of neoplastic cell proliferation will depend on a selective exploitation of these properties of 1,25D3 to design novel analogs of 1,25D3 with low hypercalcemic but high differentiation-inducing properties. It is anticipated that a better understanding of the multiple pathways that transduce the signals from 1,25D3 to the nucleus and to the cell membrane, a consideration of the structure-function relationships of the derivatives of 1,25D3, and the use of synergistic combinations of 1,25D3 with other biological response modifiers or cytotoxic drugs will lead to improved therapy of several hyperproliferative and malignant diseases.

1,25-Dihydroxy-vitamin-D3 enhances antiproliferative effect and transcription of TGF-beta1 on human keratinocytes in culture.

Both TGF-beta and 1,25-dihydroxy-vitamin-D3 (1,25(OH)2D3) have been reported to decrease the proliferation of normal human keratinocytes. The effect and expression of TGF-beta in keratinocytes treated with 1,25(OH)2D3 was investigated. Human keratinocytes were grown in the presence of various concentrations of TGF-beta and/or 1,25(OH)2D3 prior to enumeration. TGF-beta, alone, has a half maximal dose of inhibition (ED50) of approximately 750 pg/ml after seven days in culture in Keratinocyte Growth Medium (KGM; Clonetics) supplemented with 1.5 mM calcium. When 1,25(OH)2D3 (10(-7)M) was also added to cultures with various concentrations of TGF-beta, the ED50 shifted an average of 2-fold less. The presence of TGF-beta (10 pg/ml) augmented the potency of 1,25(OH)2D3 by at least 10-fold. In keratinocyte cultures, the antiproliferative effect of the two compounds together is synergistic. In keratinocytes grown for 1 week in the presence of 1,25(OH)2D3 at 10(-6)M, the TGF-beta 1 message increased approximately 5-fold. An increase is detected within 2 hours of exposure to 1,25(OH)2D3. There was only a 50% increase in the levels of TGF-beta 2 and no detection of TGF-beta 3. When keratinocyte cultures were treated with 1,25(OH)2D3 and neutralizing antibodies to TGF-beta, the induced-antiproliferative activity was blocked by more than 50%. The keratinocytes produced more active than latent TGF-beta after growth with high doses of 1,25(OH)2D3.

Direct measurement of fast axonal organelle transport in chronic ethanol-fed rats.

While indirect methods have been used, direct evaluation and measurement of the fast axonal transport system itself in chronic ethanol-fed rats have not been carried out previously. We evaluated this system using analog and digital image enhancement of differential interference contrast optical images in real time to assess the effect of ethanol on fast intra-axonal organelle traffic in rat sural nerve. A radiolabeling method for evaluating fast axonal transport was used in similar animals to compare the indirect and direct techniques. Also, the concentration of organelles (mitochondria, large and small clear vesicles, dense vesicles, and membranous whorls) in the annulospiral sensory nerve endings of muscle spindles from a foot muscle (flexor digitorum brevis) of these same animals was quantitated from electron micrographs. Rats were fed an ethanol-containing liquid diet for one to five months. Three observations were made; (1) There was no statistically significant change in the mean organelle speed in the anterograde direction, but the mean organelle speed in the retrograde direction increased 11%, 9%, and 17% (statistically significant) at 3, 4, and 5 months of ethanol exposure, respectively. (2) Significant increases in organelle content of sensory nerve endings were seen at 2 and 3 months of intoxication. (3) Increases in organelle densities in terminals were transitory and returned to normal in the face of on-going ethanol administration. We conclude: (1) There is no permanent impairment of fast axonal organelle transport in this model after 5 months of exposure. (2) Sensory endings on muscle spindles show transitory increases in organelle density. (3) Retrograde speed increases may be a partial compensatory mechanism to help restore normal terminal organelle density.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine receptor gene expression in skeletal muscle of chronic ethanol-fed rats.

The expression of the neuromuscular acetylcholine receptor (AChR) alpha-subunit gene was evaluated in soleus muscles from an animal model of chronic alcoholism. At 8 weeks of age, test rats were placed on a nutritionally complete liquid diet containing 6.7% ethanol (v/v). Age- and weight-matched control rats were pair-fed an isocaloric liquid diet. After a 16-week diet period, soleus muscles were obtained and total RNA and poly(A)+ RNA were isolated. Muscle RNA levels from ethanol-fed and control rats were comparable. AChR alpha-subunit mRNA was detected by hybridization of muscle poly(A)+ RNA with a 32P-labeled, complementary riboprobe. The steady-state level of AChR alpha-subunit mRNA was reduced by 39% (p less than 0.001) in soleus muscles from the ethanol-fed rats as compared to pair-fed controls. These results suggest that the expression of the AChR alpha-subunit gene is down-regulated after chronic ethanol exposure at a transcriptional or posttranscriptional level.

Effect of 1,25-dihydroxyvitamin D3 on human keratinocytes grown under different culture conditions.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2-D3) is known to decrease the proliferation and increase the differentiation of different cell types including human keratinocytes. The growth and differentiation of keratinocytes in the presence of 1,25-(OH)2-D3 using serum-free media formulations has been described previously. This investigation extends these studies to describe various culture conditions with human foreskin keratinocytes to determine the optimal antiproliferative activity of 1,25-(OH)2-D3. Keratinocytes were plated onto tissue culture dishes using one of three basic serum-free media protocols; a) with no feeder layer in keratinocyte growth medium (KGM); b) onto mitomycin C-treated 3T3 mouse embryo fibroblasts; or c) onto mitomycin C-treated dermal human fibroblasts. The last two protocols utilized Dulbecco's modified Eagle's Medium (DMEM) supplemented with growth factors. Keratinocyte cell growth was greatest in the KGM medium. Although the growth of keratinocytes on either feeder layer was similar, there were differences in the ability of the cells to form envelopes in the presence of 1,25-(OH)2-D3. The addition of hydrocortisone and cholera toxin to the medium also affected the response of the keratinocytes to 1,25-(OH)2-D3. The antiproliferative effect of 1,25-(OH)2-D3 was not altered by varying the extracellular calcium levels from 0.25 to 3 mM. The antiproliferative activity of 1,25-(OH)2-D3 is attenuated in cells at low density. Our results suggest that an optimal condition to investigate the ability of 1,25-(OH)2-D3 to inhibit keratinocyte proliferation is at preconfluent cell density in the presence of KGM supplemented with 1.5 mM calcium without a feeder layer. These conditions are not appropriate for investigating the enhancement of differentiation by 1,25-(OH)2-D3, but can be used to assay other agents that modulate keratinocyte proliferation.

Retrograde axonal transport in chronic ethanol-fed and thiamine-deficient rats.

Retrograde axonal transport may play an important role in the feedback regulation of protein synthesis in neuronal perikarya, and the anterograde axonal transport of protein. The "dying-back" neuropathies associated with thiamine deficiency and chronic alcoholism may arise as a consequence of altered axonal transport. We have reported alterations in fast anterograde axonal transport in rats as a result of ethanol exposure or thiamine deficiency. The present studies were undertaken to determine whether retrograde transport was also affected by these experimental treatments. One group of rats was fed a liquid diet containing 6.7% ethanol for 16 weeks. Another group of rats was made thiamine deficient with a thiamine-free diet for 4 weeks. Retrograde axonal transport was labeled by injecting the left sciatic nerve unilaterally with 3H-N-succinimidyl propionate. This compound covalently labels proteins in the nerve at the site of injection and is accumulated by retrograde axonal transport to sensory nerve cell bodies in the dorsal root ganglia and motor nerve cell bodies in the spinal cord. After 7 days, dorsal root ganglia of chronic ethanol-fed rats showed a significant 45% decrease in the amount of accumulated retrograde label compared to controls. No significant differences in accumulation were found in the spinal cords. These results suggest that the peripheral neuropathies caused by chronic alcoholism and thiamine deficiency follow different etiologies, and that motor and sensory fibers are affected differently by ethanol.

Interleukin-1 enhances epidermal wound healing.

Previous in vitro studies suggest that the interleukin-1 (IL-1) proteins may be important for skin function. Keratinocytes are known to synthesize IL-1 in response to injury and IL-1 has been shown to stimulate fibroblast and keratinocyte growth, collagen synthesis by fibroblasts, and chemotaxis of keratinocytes. In order to evaluate the ability of IL-1 to enhance wound healing, recombinant human IL-1 alpha was applied topically in a water-miscible cream to partial thickness wounds made on the backs of pathogen-free adult pigs. The wounded site was excised at various times for assessment of epidermal regeneration. This study shows a statistically significant enhancement of healing of the IL-1-treated wounds compared with either air-exposed or vehicle-treated wounds. Histologic examination of biopsies from IL-1-treated wounds indicated complete and architecturally normal epidermal regeneration. Porcine keratinocytes and membranes prepared from porcine skin were found to possess high affinity receptors for human IL-1 alpha. This study suggests that topical administration of IL-1 may be useful for the promotion of wound healing.

Temporal relationship between nerve-stump-length-dependent changes in the autophosphorylation of a cyclic AMP-dependent protein kinase and the acetylcholine receptor content in skeletal muscle.

The acetylcholine receptor (AChR) content and the autophosphorylation of the regulatory subunit of cyclic AMP-dependent protein kinase type II (R-II) were evaluated in rats soleus muscles at 24, 30 and 66 hr after surgical denervation by cutting the nerve at a short distance (short-nerve-stump) and at a long distance (long-nerve-stump) from the muscle. AChR content was based on the specific binding of [125I]alpha-bungarotoxin (BUTX); changes in the autophosphorylation of R-II were based upon the predominant in vitro 32P-phosphorylation of a 56-Kd soluble protein in cytosolic fractions of solei. The AChR content and the 32P-autophosphorylation of R-II were increased in samples from short-nerve-stump solei, but not from long-nerve-stump solei, after a denervation-time of 30 hr. This nerve-stump-length dependency indicates that the two denervation effects are not related to the immediate halt of impulse-evoked muscle contractility. Furthermore, the results show that alterations in the 32P-autophosphorylation of R-II occurred before, as well as whenever, increases in the AChR content were found. Speculatively, this temporal relationship may be significant with respect to the potential role of R-II in gene expression.

Receptors for B-melanocyte-stimulating hormone exhibit positive cooperativity in synchronized melanoma cells.

Cloudman S91 mouse melanoma cells respond in culture to B-melanocyte-stimulating hormone (B-MSH) with changes in morphology, growth rates, and melanin production. The effects of MSH appear to be mediated through a stimulation of the cyclic AMP system. It was reported earlier that at least some of the responses to MSH (increased cyclic AMP production and tyrosinase activity) occur in the G2 phase of the cell cycle [Wong, G., Pawelek, J., Sansone, M., & Morowitz, J. (1974) Nature (London) 248, 351-354] and that the apparent reason for this cell cycle restriction is that receptors for MSH are most active in the G2 phase [Varga, J. M., DiPasquale, A., Pawelek, J., McGuire, J., & Lerner, A. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 1590-1593]. In this report, we found that by two separate methods of obtaining populations of cells in the G2 phase of their cycle--centrifugal elutriation or synchronization with thymidine--we observed increased binding of MSH by cells in the G2 and possibly late S phases of their cycle. However, cultures of cells passing through their cycle in synchrony were quite different from nonsynchronized (random) cultures. Both synchronized and random cultures expressed receptors for MSH in the G2 and possibly late S phases of their cycle, but synchronized cultures bound severalfold more MSH per cell than random cultures. This increased binding of MSH by synchronized cells was accompanied by an increase in tyrosinase activity and pigment production.(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased axonal transport in rat nerve following acute and chronic ethanol exposure.

The axonal transport system, which supplies essential proteins and other cellular components to the distal portions of peripheral nerve axons, has been postulated to be the primary site of vulnerability inducing the peripheral neuropathies associated with neurotoxin exposure. Axonal transport was examined in normal rat dorsal root ganglia-sciatic nerve preparations incubated 28 hours in vitro in the presence of 79, 198 or 395 mg% ethanol. Exposure of the nerves to 395 mg% ethanol significantly reduced the accumulation of radiolabeled protein by 70%. Also, groups of rats were pair-fed an ethanol or isocaloric control diet for 9, 16 or 28 weeks. In vitro axonal transport was found to be unchanged in nerves of rats fed the ethanol diet for 9 weeks, but was significantly reduced 44% after 16 weeks and 47% after 28 weeks of ethanol feeding. These results suggest that interference with the axonal transport machinery by ethanol or perhaps acetaldehyde, its primary metabolite, may lead to the development of alcoholic peripheral neuropathy.

Energy metabolism in contracting rat skeletal muscle: adaptation to exercise training.

Rats were trained by means of a program of treadmill running. Hindlimb muscles were stimulated to contract in anesthetized rats. Measurements were made on the plantaris and the deep, predominantly fast-twitch red portion of the gastrocnemius. The concentration of ATP plus phosphocreatine (approximately P) decreased less and stabilized at a higher level, whereas inorganic phosphate (Pi) and AMP concentrations increased less and attained lower steady-state levels in trained than in untrained muscles at the same work rate. Similarly, when muscles were stimulated to contract in the perfused rat hindquarter preparation, phosphocreatine (PC) concentration decreased less in trained plantaris muscle during contractile activity that resulted in the same rate of oxygen uptake by trained and untrained muscles. In both preparations, glycogen concentration decreased less and lactate increased less in the trained muscle. From the changes that occurred in the PC-to-creatine ratio during contractile activity and from ATP concentration, it could be estimated that free ADP concentration increased less than one-half as much in trained as in untrained muscles. We conclude that, as a consequence of the adaptive increase in muscle mitochondria, approximately P concentration is higher and Pi, ADP, and AMP concentrations are lower in muscles of exercise-trained compared with untrained rats during the same contractile activity.

Role of cholinergic neuromuscular transmission in the neuroregulation of the autophosphorylatable regulatory subunit of cyclic AMP-dependent protein kinase type II and the acetylcholine receptor content in skeletal muscle.

Previously, we found that the in vitro [32P]-autophosphorylation of the regulatory subunit of cyclic AMP-dependent protein kinase type II in rat soleus muscles is subject to a nerve-stump-length-dependent neuroregulation which indicates that this event is dependent upon some neural signal other than the impulse-directed release of acetylcholine. In this investigation, tetrodotoxin and alpha-bungarotoxin were also administered to further differentiate the effect of impulse-directed and spontaneously released acetylcholine upon this event and also upon the appearance of new acetylcholine receptors as measured by the binding of radioiodinated bungarotoxin. A 24 h blockade of cholinergic transmission with either neurotoxin did not change the phosphorylation level of the regulatory subunit, while a significant increase is observed when solei are surgically denervated for this period. The phosphorylation level and also the acetylcholine receptor content were increased only after more prolonged (48-96 h) muscle inactivity was produced with the neurotoxins. However, then their effects may not be solely related to alterations in cholinergic transmission. Taken together, our results do not support a trophic role for spontaneously released acetylcholine with respect to the two neurotrophic events studied.

Increased axonal transport in peripheral nerves of thiamine-deficient rats.

Thiamine deficiency has been implicated as a significant contributing factor in the development of peripheral neuropathies in chronic alcoholic patients. We hypothesized that thiamine deficiency may lead to an alteration in axonal transport because it has been associated with "dying-back" neuropathies and its importance in neural tissue has been demonstrated with antimetabolites. To test this possibility rats were made thiamine-deficient by feeding a liquid diet lacking thiamine. Control rats were pair-fed a complete liquid diet. The deficiency developed after 3 to 4 weeks and was evidenced by anorexia, weight-loss, and a significant increase in the erythrocyte transketolase activity ratio. Also, the sural nerve conduction velocity was found to be significantly reduced in these animals (18.74 m/s) relative to that of pair-fed control rats (31.99 m/s). In vitro transport experiments utilizing dorsal root ganglia-sciatic nerve preparations indicated that twice as much [35S]methionine-labeled protein accumulated at a ligation by fast transport in the thiamine-deficient rats as in nerves of their pair-fed controls. There was no difference in the level of incorporation of radioactive precursor into the dorsal root ganglia. The increase in transport suggests that thiamine deficiency per se has no detrimental effects on the transport machinery and process, but may indicate extensive regenerative activity in the distal portions of these axons.

Decreased acetylcholine receptor content in denervated skeletal muscles infused with nerve extract.

The influence of a concentrated extract of soluble substances from the sciatic nerve upon the acetylcholine receptor (AChR) content in the soleus muscle of adult rats was examined by in vivo infusions. Internal and membrane-inserted AChR were quantitated by the specific binding of 125I-alpha-bungarotoxin (a-BuTX). Interestingly, the nerve extract had no apparent effect unless the soleus muscle was also denervated at the start of the infusion. Then, after 66 hr, substantially less (60-80%) binding of 125I-a-BuTX to AChR was observed compared to denervated solei that did not receive an infusion of nerve extract. However, the concentration of protein in the nerve extract had to exceed 5 mg/ml before this effect was evident. Infusions of phosphate-buffered saline, bovine serum albumin, rat liver extract, or human transferrin had no striking effect upon AChR. The prevention of the characteristic denervation-induced increase in non-junctional AChR by an active component in the nerve extract may be due to a trophic signal for decreased synthesis of AChR, but it is also possible that the degradation of AChR was increased.

Identification of a neuroregulated phosphoprotein in skeletal muscle as the regulatory subunit of cyclic AMP-dependent protein kinase II.

When soluble proteins in cytosolic fractions of rat soleus muscles are 32P-phosphorylated in vitro by an ATP:protein phosphotransferase reaction, the major substrate is a 56-kilodalton (56K) protein. As we have also reported previously, the onset and development of increased 32P-phosphorylation of this 56K protein, which are observed after the soleus is denervated, temporally correlate with the denervation period and length of the distal nerve stump [Held et al, 1983]. Conclusive evidence which identifies this neuroregulated muscle protein as the regulatory subunit of cyclic AMP-dependent protein kinase type II (R-II) is presented in this paper. The 56K soleus protein and purified bovine heart R-II were 32P-phosphorylated and subjected to limited proteolysis with bovine pancreas trypsin. After resolution of the generated 32P-phosphopeptides by SDS slab PAGE and visualization by autoradiography, no tryptic products were observed from the 56K soleus protein which were not also produced by proteolysis of the purified R-II. These tryptic phosphopeptides included 39, 16.5, and 12K fragments which retained the autophosphorylation site of R-II. After denervation, the 32P-phosphorylation of the 56K soleus protein and of the 39K tryptic peptide product were comparably increased. The identification of the neuroregulated 56K soleus protein as R-II was also confirmed by Western blotting with a specific anti-R-II sera. Taken together, our results demonstrate that the previously observed neuroregulation of the 32P-phosphorylation of the 56K soleus protein is identifiable with some alteration which affects the intramolecular 32P-autophosphorylation of R-II.

Phosphorylative neuromodulation of the regulatory subunit of cyclic AMP-dependent protein kinase type II in skeletal muscle.

The phosphorylative neuromodulation of the regulatory subunit of protein kinase type II (R-II) in cytosolic fractions from denervated and sham-operated, contralateral soleus muscles of the rat was evaluated. The denervation-induced increase in the 32P-phosphorylation of R-II is not related to an increased dephosphorylation by cation-dependent or cation-independent protein phosphatases in the cytosolic fractions. The level of 32P-phosphorylation of an exogenous heptapeptide substrate (Kemptide) by dissociated catalytic subunits of cyclic AMP-dependent protein kinase in cytosolic fractions from denervated and sham-operated solei did not differ. Also, no change in the concentration of cytosolic R-II assessed by competitive enzyme-linked immunosorbent assays (ELISA) was found after denervation. However, the in vitro 32P-phosphorylation of R-II in these samples was increased. Taken together, our results suggest that the increased availability of autophosphorylatable sites reflects an in vivo modulation of R-II phosphorylation rather than a significant change in total R-II content.