Cytochemical localization of adenylate cyclase activity in rat olfactory cells

Summary Adenylate cyclase activity was demonstrated in the cilia, dendritic knob and axon of rat olfactory cells by using a strontium-based cytochemical method. The activity in the cilia and the dendritic knob was enhanced by non-hydrolyzable GTP (guanosine triphosphate) analogues and forskolin, and inhibited by Ca²⁺, all in agreement with biochemical reports of the odorant-sensitive adenylate cyclase. The results support the hypothesis of cyclic AMP working as a second messenger in olfactory transduction and imply that the transduction sites exist not only in the olfactory cilia but also in the dendritic knob. Enzymatic activity was also observed in the olfactory dendritic shaft by treating the tissue with 0.0002% Triton X-100, although the properties and role of the enzyme in this region are uncertain. The detergent inhibited the enzymatic activity in the cilia and the dendritic knob.     

Cytochemical localization of adenylate cyclase in the sodium-transporting epithelium isolated from frog skin

Summary A modified cytochemical technique with 5-adenylylimidodiphosphate as substrate, was used to examine the distribution of adenylate cyclase in cells comprising the transepithelial Na⁺ transport pathway in isolated frog skin epithelium. Particular attention was paid to the effects of fixation on the activity and localization of adenylate cyclase. Fixation in glutaraldehyde alone or in combination with paraformaldehyde reduced the amount of reaction product, while better results were obtained using unfixed tissues. Optimum results were obtained following stimulation of adenylate cyclase with forskolin and in the presence of specific metabolic inhibitors. Adenylate cyclase was localized in the basolateral membranes of the principal cells which constitute a functional syncytium for Na⁺ transport and was absent from the apical membranes of the outermost granulosum cells. This distribution is consistent with the transepithelial Na⁺ transport model and defines the functional morphology of the cells involved in Na⁺ transport across frog skin. The results are compatible with the process of Na⁺ re-absorption across other epithelial cells, verifying that frog skin is a convenient model-tissue to study Na⁺ transport mechanisms. Adenylate cyclase was also found in membranes of the mitochondria-rich cells, a minor and parallel Na⁺ transporting pathway.     

Cytochemical localization of adenylate cyclase and 3',5'-nucleotide phosphodiesterase in Dictyostelium.

Cytochemical localizations of adenylate cyclase and 3′,5′-nucleotide phosphodiesterase were performed on aggregating Dictytostelium discoideum myxamoebae. The adenylate cyclase reaction product was localized on the inner surface of the plasma membrane. The phosphodiesterase reaction product was localized on the outer surface of the plasma membrane. Differences in enzyme activity were noted according to the state of cell (isolated or aggregated) and according to the cell position in larger aggregates. Heavy precipitation indicative of adenylate cyclase activity was not observed in isolated amoebae, but was often observed in streams and in some cells of aggregates. The precipitation indicative of phosphodiesterase activity could be found in isolated amoebae and in peripheral cells of aggregates.     

Cytochemical demonstration of adenylate cyclase activity with cerium

Summary Cerium was applied for the ultrastructural, cytochemical localization of adenylate cyclase (EC 4.6.1.1.). The enzyme activity was stimulated with norepinephrine, prenalterol and choleratoxin in the brown fat cells of newborn rats. The final reaction product was observed in the plasmalemmas of the stimulated adipocytes. The precipitate was finely crystalline, easily visible in the electron microscope and in the X-ray microprobe analysis it yielded cerium and phosphate peaks, respectively. The use of cerium offers a new tool valid for the cytochemical localization of adenylate cyclase enzyme related to the membrane receptors.This study was supported by the grant from Reino Lahtikari Foundation     

Cytochemical demonstration of adenylate cyclase activity with cerium

Cerium was applied for the ultrastructural, cytochemical localization of adenylate cyclase (EC 4.6.1.1.). The enzyme activity was stimulated with norepinephrine, prenalterol and cholera toxin in the brown fat cells of newborn rats. The final reaction product was observed in the plasmalemmas of the stimulated adipocytes. The precipitate was finely crystalline, easily visible in the electron microscope and in the X-ray microprobe analysis it yielded cerium and phosphate peaks, respectively. The use of cerium offers a new tool valid for the cytochemical localization of adenylate cyclase enzyme related to the membrane receptors.     

Subcellular localization and partial characterization of bovine corpus luteum adenylate cyclase.

The subcellular localization of adenylate cyclase (ATP pyrophosphatelyase (cyclizing), EC 4.6.1.1) in bovine corpus luteum was studied using isotonic and hypotonic homogenization and fractionation conditions. All fractions prepared were assayed for adenylate cyclase, marker enzymes and DNA. Only plasma membrane marker enzyme, 5'-nucleotidase paralleled the distribution of adenylate cyclase under both isotonic and hypotonic conditions (conditionsoth isotonic and hypotonic conditions (coefficient of correlation = 0.95). Two main fractions prepared under hypotonic conditions were subfractionated by discontinuous sucrose gradient centrifugation. The highest amount of adenylate cyclase was found in a fraction having a density approximately equal to 1.13 g/cm3. The specific activity of this fraction was 4--6 times higher than that of the homogenate. The electron microscopic study of this fraction revealed the presence of a single type of particulate material consisting of small vesicles exhibiting a typical unit membrane structure. It is concluded that this adenylate cyclase is primarily localized in the plasma membranes. Basal adenylate cyclase activity of plasma membranes was stimulated 2--3 times by luteinizing hormone (10 mug/ml), 3--4 times by prostaglandin E2 (10 mug/ml), 4--6 times by NaF (0.01 M) and two times by methanol (0.2%).     

Cytochemical localization of adenylate cyclase in the various tissues of Locusta migratoria (migratorioides R.F.)

Summary The ultrastructural cytochemical procedure to demonstrate adenyl cyclase in mammalian organs was used in insects. After several modifications, an utilizable method was applied for the detection of the enzyme in the various tissues. Adenylate cyclase which can be stimulated with octopamine was localized on the membrane of the glial cells and the axolemma of certain large axons in the insect brain. Adenylate cyclase which could be activated by NaF and isoproterenol was also demonstrated in the lipid droplets of glial cells of the brain. With the simultaneous application of NaF and isoproterenol, rather strong adenylate cyclase activity could be detected on the surface of the corpora allata cells both in the cells situated on the glandular surface and the central part of the gland. In contrast in the corpus cardiacum enzyme activity was only observable on the basal lamina of the glandular surface. An appreciable amount of reaction product, indicating the presence of the enzyme, could be found on the surface of the lipid droplets in the fat body situated near the glandular tissues. In the heart muscle, reaction product referring to enzyme activation could not be demonstrated with the help of the methods applied.     

Cytochemical localization of K+-dependent p-nitrophenyl phosphatase and adenylate cyclase by using one-step method in human washed platelets

Summary Ultrastructural cytochemical localization of ouabain-sensitive, potassium dependent p-nitrophenyl phosphatase (K⁺-NPPase) of the Na⁺-/K⁺-ATPase complex and adenylate cyclase (cAMPase) activities, in washed inactivated human platelets, are described. The one-step lead-citrate method, under similar incubation conditions, was used to determine both activities. K⁺-NPPase appeared in both plasma membrane and the surface-connected canalicular system (SCCS) of the platelets. These data suggest a uniform distribution of the enzyme throughout membrane systems which are in contact with the external medium. cAMPase activity was strictly localized in tubules of the dense tubular system (DTS) when incubation medium contained prostaglandin E₁, prostaglandin D₂ or forskolin, at concentrations known to stimulate the enzyme in platelets that are intact. This fact and the inhibition of cytochemical reaction by thrombin confirm that the one-step lead-citrate method is a useful procedure in determining adenylate cyclase, abolishing the unfavorable conditions of previously reported methods.     

Cytochemical demonstration of adenylate cyclase with strontium chloride in the rat pancreas

A cytochemical procedure for the localization of adenylate cyclase with Sr2+ as the capture ion and adenylyl imidodiphosphate as the specific substrate was evaluated in the rat pancreas. Incubation medium was unaffected by the addition of 5 mM strontium ions but became turbid in the presence of lead or strontium plus 10 mM NaF. Tissues were prefixed in 2% formaldehyde/0.5% glutaraldehyde and incubated, and the cytochemical precipitate was converted to the Pb2+ salt. Enzymatic activity was demonstrated on the plasma membrane of pancreatic acinar cells and responded to stimulation by secretin. Controls frequently contained Pb2+ sequestered in mitochondria, but otherwise only a few randomly distributed grains were observed. The controls were 1) omission of substrate from the medium; 2) incubation of tissue for 1 min in complete medium; and 3) tissue previously inactivated by microwave irradiation and incubated for 30 min in complete medium including secretin.     

Cytochemical studies of myocardial adenylate cyclase after its activation and inhibition

Incubation medium, as previously described (J Histochem Cytochem 27:774, 1979), was used to demonstrate the presence of adenylate cyclase (AC) in myocardium. NaF and ouabain were used to inhibit adenosine triphosphatases (ATP) and NaF and isoproterenol were used as activators of AC. The inhibitory effect of adenosine on AC was blocked by the addition of adenosine deaminase. The addition of tetramisol blocked the influence of the alkaline phosphatases on adenylyl imidodiphosphate hydrolysis. The use of these substances resulted in specific precipitation localized in junctional sarcoplasmic reticulum and sarcolemma. The reaction product was dramatically intensified after activation of AC by NaF or isoproterenol. Preincubation in 10-100 mM of propranolol, for 30 min, blocked AC stimulation by isoproterenol and prevented the appearance of the specific precipitate. The localization of specific precipitate in junctional sarcoplasmic reticulum and subsarcolemmal cisternae corresponds to the localization of Na+, K+ ATPase and may reflect the similar role that AC and Na+, K+ ATPase play in calcium release from sarcoplasmic reticulum of internal and peripheral couplings.     

Subcellular localization of adenylate cyclase of buffalo spermatozoa

The intracellular localization of adenylate cyclase and 3',5'-cyclic nucleotide phosphodiesterase in buffalo sperm was examined. Adenylate cyclase activity is distributed in heads (8.4%), midpieces (16.6%), tails (49.5%) and 5.7% in the soluble supernatant; the total recovery being 81%. A 4-fold increase in specific activity was observed in the tail fraction relative to sonicated suspension. Further fractionation of the tail fraction into plasma membrane and microtubules by dialysis against low ionic strength buffer was followed by marker enzymes (Mg2+ -ATPase, 5'-nucleotidase and alkaline phosphatase) as well as by examination of fractions under electron microscope. The recovered adenylate cyclase (79%) was found in microtubules (45%) and plasma membrane (34%). Cyclic nucleotide phosphodiesterase in tails was distributed in tail plasma membrane (13.7%), microtubules (31.5%) and cytosol (34%) with a total recovery of 80%. Similar results were obtained when the distribution of adenylate cyclase and cyclic nucleotide phosphodiesterase was studied by treatment with Triton X-100; 40% activity of adenylate cyclase present in tails (about 20% relative to sperm sonicate) appeared in the soluble form by this method. The results are discussed in relation to control of cyclic AMP levels in buffalo sperm by adenylate cyclase and cyclic nucleotide phosphodiesterase.     

Ultracytochemical localization of adenylate cyclase activity in rat thymocytes

Summary Ultrastructural localization of adenylate cyclase (AC) activity was investigated in suspensions of unfixed isolated rat thymocytes using a medium containing 0.6 mM 5-adenylylimidodiphosphate (AMP-PNP) as a substrate, 10 mM MgSO₄ as an activator, 5 mM theophylline as an inhibitor of 3,5-AMP-phosphodiesterase and 2 mM lead nitrate as a capturing agent. AC activity was demonstrated in plasma membrane, perinuclear space, endoplasmic reticulum, Golgi complex, centriole microtubules and mitochondria. AC was activated with 10^–4 M adrenalin in the presence of 5-guanylylimido-diphosphate (GMP-PNP) as well as with 10^–2 M NaF. In the cells incubated in a medium devoid of theophylline and containing 5-AMP instead of AMP-PNP, 5-nucleotidase activity was observed in the same cell structures as AC activity. Hydrolysis of 5-AMP in the nucleus was much stronger than that of AMP-PNP. 10 mM NaF markedly inhibited hydrolysis of 5-AMP in all cell structures. No staining was observed with 2 mM -glycerophosphate as a substrate. Incubation of unfixed thymocytes in media containing AMP-PNP, 5-AMP or p-nitrophenyl phosphate, but not -glycerophosphate, induced both in the nucleus and in the cytoplasm in some cells an appearance of a transitory reticular formation consisting of about 30 nm thick strands which could penetrate the nuclear envelope and plasma membrane and form connections with adjacent cells. The transitory reticular formation seems to belong to the cytoskeleton and to be involved in cell aggregation.     

Ultrastructural localization of adenylate cyclase activity in chicken osteoclasts

Using lead citrate as a capture reagent and adenylate-(beta, gamma-methylene) diphosphate (AMP-PCP) as a substrate, we localized adenylate cyclase activity on the non-ruffled border plasma membrane of approximately half of the osteoclasts on trabecular bone surfaces in the tibial metaphyses of chickens fed a low (0.3%)-calcium diet. The enzyme was not detectable in osteoclasts when chickens were fed a normal calcium diet. Activity was observed on the entire plasma membrane of detached osteoclasts that were situated between osteoblasts on the bone surface and blood vessels in the marrow cavity. Detection of activity on detached osteoclasts required the presence of an activator, implying lower levels in these cells than in those with ruffled borders. Staining was greater on the lateral sides of osteoblasts and osteoclasts when they were in contact with each other. Reaction specificity was indicated by the demonstration of stimulation by forskolin, guanylate-(beta, gamma-methylene) diphosphate (GMP-PCP), dimethylsulfoxide, and NaF, inhibition by alloxan and 2',5'-dideoxyadenosine, and absence of activity when sections were incubated in substrate-free medium or when GMP-PCP replaced AMP-PCP as a substrate. The finding of adenylate cyclase in osteoclast plasma membrane provides structural evidence that the adenylate cyclase-cyclic AMP system has a role in regulation of osteoclast cell function. The low-calcium diet appears to have resulted in increased amounts of adenylate cyclase in osteoclasts.     

Cytochemical localization of K(+)-dependent p-nitrophenyl phosphatase and adenylate cyclase by using one-step method in human washed platelets.

Ultrastructural cytochemical localization of ouabain-sensitive, potassium dependent p-nitrophenyl phosphatase (K(+)-NPPase) of the Na(+)-/K(+)-ATPase complex and adenylate cyclase (cAMPase) activities, in washed inactivated human platelets, are described. The one-step lead-citrate method, under similar incubation conditions, was used to determine both activities. K(+)-NPPase appeared in both plasma membrane and the surface-connected canalicular system (SCCS) of the platelets. These data suggest a uniform distribution of the enzyme throughout membrane systems which are in contact with the external medium. cAMPase activity was strictly localized in tubules of the dense tubular system (DTS) when incubation medium contained prostaglandin E1, prostaglandin D2 or forskolin, at concentrations known to stimulate the enzyme in platelets that are intact. This fact and the inhibition of cytochemical reaction by thrombin confirm that the one-step lead-citrate method is a useful procedure in determining adenylate cyclase, abolishing the unfavorable conditions of previously reported methods.