Lymphocyte surface modulation and cyclic nucleotides I. Topographic correlation of cyclic adenosine 3':5'-monophosphate and immunoglobulin immunofluorescence during lymphocyte capping.

The cross-linking of human peripheral lymphocyte surface Ig results in an early association of cyclic adenosine 3':5'-monophosphate (cAMP) and the cell surface Ig patches. Examination of the subsequent stages of cap formation reveals the continued relationship of cAMP and the clustered surface Ig. In addition, the generalized influx of calcium produced by the ionophore A-23187 disrupts human lymphocyte caps. During the process of cap dissolution cAMP is still associated with surface Ig. Therefore, it is hypothesized that the localized concentration of cyclic nucleotide and calcium ion regulates the movement of cell surface constituents by coordinating the function of the cell's contractile and structural elements.

Localization of the catalytic subunit of cyclic AMP-dependent. Protein kinase in cultured cells using a specific antibody.

We developed a specific antibody to the catalytic subunit (C-subunit) of cyclic AMP-dependent protein kinase and used it to localize C-subunit in cultured cells. C-subunit antigen was purified from bovine cardiac muscle and cross-linked to hemocyanin with glutaraldehyde. Immunized goat serum showed a low titer of antibody after boosting; it was enriched 100-fold by affinity chromatography on catalytic subunit-Sepharose. The antibody immunoprecipitated C-subunit from type I and type II holoenzyme and depleted enzymatic activity from solution. At 12.5 nM antigen, 1 microgram antibody immunoprecipitated 10 ng of C-subunit. Immunoprecipitation of 35S-labeled cell extracts and 125I-antibody detection on nitrocellulose paper revealed that the antibody specifically reacts with C-subunit in Chinese hamster ovary (CHO) whole cell extracts. Using indirect immunofluorescence to localize C-subunit, we found a pattern of diffuse staining in the cytoplasm of CHO cells with little or no nuclear staining. A similar distribution of the enzyme was observed in Swiss 3T3 cells, bovine endothelial tracheal cells, human lung fibroblasts and NRK cells. Treatment of CHO cells with 8-bromo-cyclic AMP produced no change in the pattern or intensity of immunofluorescence. We conclude that the majority of C-subunit is localized in cytoplasm and that in cultured fibroblasts exposure to cyclic AMP analogues causes no apparent redistribution of catalytic subunit.

Immunofluorescent localization of cyclic nucleotide-dependent protein kinases on the mitotic apparatus of cultured cells.

Cyclic nucleotides and cyclic nucleotide-dependent protein kinases have been implicated in the regulation of cell motility and division, processes that depend on the cell cytoskeleton. To determine whether cyclic nucleotides or their kinases are physically associated with the cytoskeleton during cell division, fluorescently labeled antibodies directed against cyclic AMP, cyclic GMP, and the cyclic nucleotide-dpendent protein kinases were used to localize these molecules in mitotic PtK1 cells. Both the cyclic GMP-dependent protein kinase and the type II regulatory subunit of the cyclic AMP-dependent protein kinase were localized on the mitotic spindle. Throughout mitosis, their distribution closely resembled that of tubulin. Antibodies to cyclic AMP, cyclic GMP, and the type I regulatory and catalytic subunits of the cyclic AMP-dependent protein kinase did not label the mitotic apparatus. The association between specific components of the cyclic neucleotide system and the mitotic spindle suggests that cyclic nucleotide-dependent phosphorylation of spindle proteins, such as those of microtubules, may play a fundamental role in the regulation of spindle assembly and chromosome motion.

Localization of cyclic GMP and cyclic AMP in cardiac and skeletal muscle: immunocytochemical demonstration.

When rat cardiac and skeletal muscle are explored by immunocytochemical procedures designed to show sites of localization of adenosine 3',5'-monophosphate (cyclic AMP) and guanosine 3',5'-monophosphate (cyclic GMP), distinct staining patterns for the two nucleotides are seen. Antibody to cyclic AMP is found in the area of the sarcoplasmic reticulum, while antibody to cyclic GMP is found with a periodic distribution corresponding to that of the A band. This suggests a role for cyclic GMP in the regulation of myosin.

Compartmentalization of cyclic AMP during phagocytosis by human neutrophilic granulocytes.

Immunocytochemistry shows that early during phagocytosis of zymosan, adenosine 3',5'-monophosphate (cyclic AMP) appears on the cell surface before the phagosome is internalized. The appearance of cyclic AMP on the cell surface is coincident with that of granule products and regulatory subunit of type I cyclic AMP-dependent protein kinase. Guanosine 3',5'-monophosphate is not associated with the initiation site of phagocytosis, but is observed throughout the granular cytoplasmic region. This sharply localized accumulation of cyclic AMP may serve as a signal for the initiation of phagocytosis.

Neurotransmitters increase cyclic nucleotides in postganglionic neurons: immunocytochemical demonstration.

Dopamine increases adenosine 3',5'-monophosphate (cyclic AMP) but not guanosine 3',5'-monophosphate (cyclic GMP) in slices of bovine sympathetic ganglion; this increase is localized to the postganglionic neurons. Conversely, acetylcholine increases cyclic GMP but not cycle AMP in the ganglion; this increase also occurs within postganglionic neurons. Thus, different neurotransmitters can selectively alter cyclic nucleotide levels within the same neuronal population.

Cyclic guanosine and adenosine 3',5'-monophosphates in canine thyroid: localization by immunofluorescence.

When guanosine 3',5'-monophosphate (cyclic GMP) and adenosine 3',5'-monophosphate (cyclic AMP) are localized in canine thyroid by a flurescence Immunocytochemical procedure, distinct staining patterns for each nucleotide are seen: Cyclic AMP is distributed throughout the follicular cell cytoplasm before and after administration of thyroid-stimulating hormone, while cyclic GMP is localized to the follicular cell mumbrane in the control state, and increased cytoplasmic fluorescence is visualized after acetylcholine. These data provide histological evidence that correlates with cyclic nucleotide tissue measurements, sugesting diverse roles of the two nucleotides in thyroid function.

Noradrenergic stimulation of cyclic adenosine monophosphate in rat Purkinje neurons: an immunocytochemical study.

A specific immunofluorescent histochemical method for cyclic adenosine monophosphate was used to study rat cerebellum. After topical treatment with norepinephrine or stimulation of norepinephrine-containing afferents from locus coeruleus, there was a striking increase in the number of Purkinje cells with strong cyclic adenosine monophosphate reactivity. Other putative inhibitory transmitters had no significant effect on staining of Purkinje cells. The results provide the first histochemical support for the hypothesis that cyclic adenosine monophosphate can be generated postsynaptically in central neurons in response to noradrenergic stimuli.

Adenosine 3',5'-monophosphate is localized in cerebellar neurons: immunofluorescence evidence.

Adenosine 3',5'-monophosphate is localized in specific cerebellar neurons, as shown by fluorescence immunocytochemistry with a specific rabbit immunoglobulin. Positive staining is exhibited by Purkinje neurons and granule cells. The increase in concentration of cyclic adenosine monophosphate in the cerebellum, which is known to follow decapitation, is represented by greatly increased fluorescence of Purkinje neurons only. These immunofluorescence data provide the first evidence for localization of cyclic adenosine monophosphate in specific neurons and may permit further exploration into the role of this cyclic nucleotide in neuronal function.

Plasma clearance rates and renal clearance of 3H-labeled cyclic AMP and 3H-labeled cyclic GMP in the dog.

Previously, in an attempt to understand the mechanisms involved in the regulation of plasma cyclic nucleotides, we measured concentrations of adenosine 3',5'-monophosphate (cAMP) and guanosine 3',5'-monophosphate (cGMP) in plasma from selected blood vessels of anesthetized dogs. The observation that the renal venous plasma concentrations of both cyclic nucleotides were less than arterial concentrations suggested that the kidney might be an important site for the elimination of these compounds from plasma and prompted further investigation of the renal handling of these compounds. Tracer doses of either [(3)H]cAMP or [(3)H]cGMP were administered to anesthetized dogs by constant intravenous infusion, and metabolic clearance rates were determined. Concentrations of endogenous cyclic nucleotide and of cyclic nucleotide radioactivity were measured in aortic and renal venous plasma as well as in urine. Renal venous plasma [(3)H]cGMP was 39% and [(3)H]cAMP was 65% of the concentration in arterial plasma. Endogenous cyclic nucleotide levels showed a similar relationship. The plasma clearance rates (PCR) were 271+/-27 ml/min (mean+/-SE) for cGMP and 261+/-17 for cAMP. The total kidney clearance (calculated as the renal plasma flow x renal cyclic nucleotide extraction ratio) accounted for 52+/-4% and 30+/-2% of the PCR for cGMP and cAMP, respectively. Only about two-thirds of the total kidney clearance of each cyclic nucleotide could be accounted for by urinary excretion, the remainder presumably being the result of renal metabolism. The urinary clearances of (3)H-labeled cGMP (40.9+/-4.2 ml/min) and endogenous cGMP (45.0+/-2.3 ml/min) were not significantly different from each other. Both were approximately 50% greater than the glomerular filtration rate, which was 27.1+/-2.0 ml/min, indicating that a significant amount of urinary cGMP is derived from plasma by tubular secretion. In contrast, the urinary clearances of (3)H-labeled cAMP (23.7+/-1.9 ml/min) and endogenous cAMP (27.2+/-2.6 ml/min) were nearly equal both to each other and to the glomerular filtration rate, which was 24.6+/-1.7 ml/min. Thus, in the dog, glomerular filtration of plasma cAMP appears to be responsible for most of the cAMP found in urine. Renla production of cAMP, which in humans contributes from a third to a half of the urinary cAMP, was quantitatively of minor importance in the dog.Thus, under the conditions of these experiments in dogs, renal elimination appears to be responsible for half of the PCR of cGMP and about a third of the PCR of cAMP. About a third of the renal elimination of both cyclic nucleotides appears to be due to metabolic degradation within the kidney, and the balance is due to excretion in the urine.

Sources of cyclic nucleotides in plasma.

In order to determine the sites of net production and removal of the cyclic nucleotides in plasma, various blood vessels were catheterized in 17 anesthetized dogs and arterial and venous concentrations of adenosine 3',5'-monophosphate (cAMP) and guanosine 3',5'-monophosphate (cGMP) were measured by radioimmunoassay. Aortic cAMP was 30+/-2 nM (mean+/-SE) and cGMP was 13+/-1 nM. There were no significant differences for either cyclic nucleotide between the concentration in the aorta and that in the inferior vena cava, coronary sinus, hepatic vein, and femoral vein. The concentration of cAMP in renal venous plasma was 25% lower than in aortic plasma, and renal venous cGMP was 51% lower than in the aorta. The pulmonary arterial concentrations of cAMP and cGMP were slightly lower than in the aorta. The concentration of cGMP in the superior mesenteric vein plasma was 83% greater than in aortic plasma; the concentration of cAMP in this vessel was only 16% greater than that in the aorta. Superior vena cava concentrations of both cyclic nucleotides were slightly greater than arterial concentrations. THE RESULTS SUGGEST THAT: (a) the kidneys are a major site of removal of both cyclic nucleotides from plasma. (b) The lungs may be a site of net addition of both cyclic nucleotides to plasma. (c) The small intestine is a site of net production of both cyclic nucleotides, particularly cGMP. (d) The liver probably removes cyclic nucleotides from plasma. (e) Since no other organs or regions studied added detectable net amounts of cyclic nucleotides to plasma, and since the turnover of these compounds in plasma is known to be rapid, the production of plasma cyclic nucleotides under basal conditions may well be the result of small net contributions may well be the result of small net contributions from many tissues or bidirectional fluxes between tissues and plasma, or both.

Human lymphocytic metabolism. Effects of cyclic and noncyclic nucleotides on stimulation by phytohemagglutinin.

The effects of extracellular nucleotides and agents which elevate intracellular cyclic adenosine 3',5'-monophosphate (cyclic AMP) concentrations on human lymphocyte metabolism have been studied. Aminophylline, isoproterenol, and prostaglandins, all of which elevate lymphocyte cyclic AMP levels, inhibited incorporation of (3)H-labeled thymidine, uridine, and leucine into the DNA, RNA, and protein of phytohemagglutinin (PHA)-stimulated lymphocytes. Aminophylline inhibition was maximal only when the inhibitor was added within 1 hr after exposure of cells to PHA, suggesting that a relatively early step in the lymphocyte transformation process may be affected. The addition of various nucleotides to the culture medium also inhibited incorporation of labeled precursors. The best inhibitor, dibutyryl cyclic AMP (DU cyclic AMP), produced maximal inhibition only if present during the 1st hr after initial exposure to PHA. Among the various cyclic nucleotides derivatives of guanosine and adenine were the most effective inhibitors (substantial inhibition at 0.1 mM concentrations). However, the inhibition was not specific for nucleotides containing the cyclic phosphodiester moiety since the tri-, di-, and monophosphates of adenosine and guanosine were equally effective in diminishing thymidine uptake. The above inhibitions were not due to secondary effects of the inhibitors on the interaction of PHA with lymphocytes as judged by (125)I-labeled PHA binding studies.Low concentrations (1-10 mumoles/liter) of cyclic AMP produced slight stimulation of thymidine-(3)H uptake in resting lymphocytes (lymphocytes not stimulated with PHA). However, the effects were quite small as compared with those produced by PHA itself. Attempts to demonstrate increased thymidine uptake 48 hr after pulsing lymphocytes with aminophylline or isoproterenol were unsuccessful. The relationship of these observations to a possible regulatory role for cyclic AMP in PHA-stimulated lymphocytes is discussed.

Tissue guanosine-3',5'-cyclic monophosphate levels and soluble guanylate cyclase activity: a positive correlation during unilateral cryptorchidism in the rat testis.

The relationship between the subcellular distribution of guanylate cyclase and tissue guanosine-3',5'-cyclic monophosphate (cGMP) levels was investigated in rat testes after surgically induced unilateral cryptorchidism. Placement of one of a testis pair in the abdominal cavity results in loss of testicular weight and function in the abdominal testis whereas the remaining scrotal testis appears to be functionally normal. Within 5 days after surgery, tissue cGMP levels were increased by twofold in the abdominal testis. A fourfold elevation was noted from 10 to 30 days after surgery. Whereas the homogenate guanylate cyclase activity was only slightly elevated 10 and 20 days postoperatively, a 200% increase in the soluble guanylate cyclase activity was seen at 5 days. Between 10 and 30 days, the rise in activity was >250% (P < 0.01). An increase in soluble guanylate cyclase activity was noted when the data were expressed as per milligram protein, per milligram DNA or per whole testis. Conversely, particulate guanylate cyclase activity was reduced by 40% in the cryptorchid testis. Kinetic analysis of the soluble enzyme prepared from abdominal and scrotal testes yielded linear Line-weaver-Burke plots for both enzyme preparations with an identical K(m) for guanosine triphosphate, but a three-fold higher maximal velocity for the abdominal enzyme. When the soluble guanylate cyclases from both testes were mixed and assayed together, the activities were additive rather than exhibiting synergism or inhibition. These experiments indicate that the altered V(max) is not due to a transferable activator or inhibitor.An immunocytochemical technique was used to assess the cell type in which the alterations in cGMP metabolism occurred. Comparison of the scrotal and abdominal testes revealed that the abdominal testis exhibited enhanced cGMP immunofluorescence within the cells lining the inner aspect of the seminiferous tubule as well as tubular elements and interstitial cells. Thus, it is inferred that the correlated changes in soluble guanylate cyclase activity and cGMP levels occur in several of the cell types that remain viable within the cryptorchid testis.

Cyclic adenosine 3',5'-monophosphate in human lymphocytes. Alterations after phytohemagglutinin stimulation.

We have studied cyclic adenosine 3',5'-monophosphate (cyclic AMP) concentrations in human peripheral blood lymphocytes after stimulation with phytohemagglutinin (PHA), isoproterenol, prostaglandins, and aminophylline. Purified lymphocytes were obtained by nylon fiber chromatography, and low speed centrifugation to remove platelets. Cyclic AMP levels were determined by a highly sensitive radioimmunoassay. At concentrations of 0.1-1.0 mmoles/liter isoproterenol and aminophylline produced moderate increases in cyclic AMP concentrations, whereas prostaglandins produced marked elevations. High concentrations of PHA produced 25-300% increases in cyclic AMP levels, alterations being demonstrated within 1-2 min. The early changes in cyclic AMP concentration appear to precede previously reported metabolic changes in PHA-stimulated cells. After 6 hr cyclic AMP levels in PHA-stimulated cells had usually fallen to the levels of control cells. After 24 hr the level in PHA-stimulated cells was characteristically below that of the control cells. Adenyl cyclase, the enzyme which converts ATP to cyclic AMP, was measured in lymphocyte homogenates. Adenyl cyclase activity was rapidly stimulated by fluoride, isoproterenol, prostaglandins, and PHA. Since adenyl cyclase is characteristically localized in external cell membranes, our results are consistent with an initial action of PHA at this level.

Control and localization of rat adrenal cyclic guanosine 3', 5'-monophosphate. Comparison with adrenal cyclic adenosine 3', 5'-monophosphate.

Cyclic AMP and cyclic GMP were measured in rat adrenal glands after either hypophysectomy alone or after hypophysectomy and treatment with ACTH. Adrenal cyclic GMP levels rise in acutely hypophysectomized rats to a maximum at 1 h of approximately 200% of control levels; there is a return to base line at 4-12 h after hypophysectomy. In contrast, adrenal cyclic AMP falls immediately to about 50% of control levels after hypophysectomy and remains at approximately 1 pmol per mg tissue. Doses of ACTH beyond the physiological range markedly suppress adrenal cyclic GMP while producing a 50-fold or greater rise in cyclic AMP in hypophysectomized rats. This pattern of adrenal cyclic GMP rise was unchanged in acutely hypophysectomized animals treated with desamethasone. N-6-2'-0 dibutyryl cyclic AMP acted similarly to the effect of ACTH in bringing about a suppression of adrenal cyclic GMP levels. Physiological i.v. pulse doses of ACTH produced a rapid dose related increase in adrenal cyclic GMP. In vitro incubation of quartered adrenal pairs with 500 mU ACTH produced elevated cyclic AMP levels and suppression of cyclic GMP. Whereas adrenal cyclic AMP fell rapidly to 50% of control levels after hypophysectomy and remained at about 1 pmol per mg tissue for 7 days, adrenal cyclic GMP showed a biphasic rhythm in long-term hypophysectomized animals. After an initial peak at 1 h after hypophysectomy, adrenal cyclic GMP declined to baseline at 4-12 h but thereafter progressively rose with time, eventually reaching levels over 1 pmol per mg tissue. Fluorescent immunocytochemical staining of rat adrenal zona fasciculata showed cyclic AMP largely confined to cytoplasmic elements with little fluorescence contained in nuclei. In constant, cyclic GMP was found discretely positioned in nuclei with prominent fluorescence in nucleoli in addition to cytoplasmic localization. It is concluded that in hypophysectomized rats ACTH, either directly or in conjunction with altertion of adrenal cyclic AMP, appears to be one factor which regulates adrenal cyclic GMP. The direction of cyclic GMP change and the different subcellular localization of the nucleotides suggest divergent roles for cyclic AMP and cyclic GMP in adrenocortical function. Furthermore, our observations suggest a role for adrenal cyclic GMP in nuclear directed events.

Effect of retinoic acid on cellular content and human parathyroid hormone activation of cyclic adenosine 3':5'-monophosphate-dependent protein kinase isoenzymes in clonal rat osteogenic sarcoma cells.

Pretreatment with 10(-8) M retinoic acid for 4 days caused changes in three distinct components of the parathyroid hormone (PTH)-stimulated cyclic adenosine 3':5'-monophosphate response in a clonal rat osteogenic sarcoma cell line, UMR 106-06: the amplitude of the cyclic adenosine 3':5'-monophosphate response to PTH was moderately increased after pretreatment with retinoic acid; while the cellular content of the two isoenzymes of the cyclic adenosine 3':5'-monophosphate-dependent protein kinase was approximately equal in control cells, retinoic acid pretreatment was associated with a marked increase in the ratio of type II to type I holoenzyme activity. This change might be due to a decrease in the type I holoenzyme as suggested by immunofluorescence detection of decreased type I regulatory subunit in fixed cells together with the relative decrease in type I holoenzyme determined biochemically; there was a marked alteration of the pattern of PTH-stimulated protein kinase isoenzyme activation from predominantly type I isoenzyme in control cells to almost exclusively type II isoenzyme in retinoic acid-treated cells. Growth inhibition by submaximal amounts of PTH and retinoic acid when added together was greater than that for either agent alone.

Particulate guanylate cyclase of skeletal muscle: effects of Ca2+ and other divalent cations on enzyme activity.

The properties of particulate guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) from purified rabbit skeletal muscle membrane fragments were studied. Four membrane fractions were prepared by sucrose gradient centrifugation and the fractions characterized by analysis of marker enzymes. Guanylate cyclase activity was highest in the fraction possessing enzymatic properties typical of sarcolemma, while fractions enriched with sarcoplasmic reticulum had lower activities. In the presence of suboptimal Mn2+ concentrations, Mg2+ stimulated particulate guanylate cyclase activity both before and after solubilization in 1% Triton X-100. Guanylate cyclase activity was biphasic in the presence of Ca2+. Increasing the Ca2+ concentration from 10(-8) to 10(-5) M decreased the specific activity. As the Ca2+ concentration was further increased to 5 . 10(-4) M enzyme activity again increased. After solubilization of the membranes in 1% Triton X-100, Ca2+ suppressed enzyme activity. Studies utilizing ionophore X537A indicated that the altered effect of Ca2+ upon the solubilized membranes was independent of asymmetric distribution of Ca2+ and Mg2+.

Characterization of rat testicular guanylate cyclase during development.

The biochemical characteristics of rat testicular guanylate cyclase were investigated and the activity and subcellular distribution of the enzyme was determined during testicular development. Examination of the effects of metal ions, nucleotides, detergents and other in vitro activators on the activity of guanylate cyclase revealed that the testicular enzyme is similar in most respects to guanylate cyclase isolated from other mammalian tissues. Changes in the total activity of guanylate cyclase during testicular development paralleled changes in the tissue concentration of cyclic GMP; i.e. guanylate cyclase activity and tissue cyclic GMP were highest during the early stages of development. Subcellular fractionation revealed that the activity of the soluble form of guanylate cyclase was best correlated with tissue cyclic GMP. Biochemical analysis of the soluble enzyme prepared from testes of neonatal and adult rats did not reveal any significant differences in the characteristics of the enzyme during ontogeny with the exception of a 2.5 fold increase in V noted in the neonatal testis. The results of this study are consistent with a molecular mechanism that allows independent regulation of the different forms of guanylate cyclase.

Cyclic nucleotides in pancreatic islets. Tolbutamide- and arginine-induced insulin release.

With the use of isolated rat islet perfusion, levels of the islet cyclic adenosine 3' ,5' -monophosphate (cAMP) were compared with dynamic insulin secretion induced by tolbutamide and arginine. Tolbutamide elevated islet cAMP rapidly and augmented both glucose-induced islet cAMP levels and insulin secretion; arginine, however, did not elevate islet cAMP but did enhance glucose-induced insulin secretion. Since the latter result could have been modulated by cyclic guanosine 3' ,5' -monophosphate, this cyclic nucleotide was also measured and found to remain unchanged during stimulation of insulin secretion by arginine and a combination or arginine and glucose. Thus, the action of tolbutamide appears to be modulated in part by cAMP, whereas arginine appears to augment insulin secretion independently of cyclic nucleotides.

Immunocytochemical localization of cyclic GMP, cGMP-dependent protein kinase, calmodulin and calcineurin in Paramecium tetraurelia.

The localization of cGMP, cGMP-dependent protein kinase, calmodulin and the calmodulin-binding protein calcineurin in Paramecium tetrauelia cells has been examined with immunocytochemical methods. These molecules appeared to be localized to a large extent in the cilia of this protozoan. To ascertain that antibodies had access to all cellular compartments we have used three different preparations for immunocytochemistry: (i) with 'whole cell' preparations immunofluorescent staining for the four molecules was mainly visible in the cilia; (ii) in 'deciliated' Paramecium, staining for cGMP and calmodulin was found in regular patterns on the cell surface most likely representing kinetosomes; (iii) using 'sectioned cells', additional cytoplasmic calmodulin appeared to be associated with glycogen particles as evidenced by the disappearance of the granular staining pattern after preincubation with alpha-amylase. In contrast, cGMP, cGMP-dependent protein kinase and calcineurin fluorescence was only very weak and diffuse in cell bodies. No nuclear fluorescence was detectable after staining with any of the antibodies. Because of the colocalization of cGMP, cGMP-dependent protein kinase, a guanylate cyclase-calmodulin-complex, and calcineurin in cilia from Paramecium, an involvement of these components in the regulation of ciliary activity is discussed.