Does histamine stimulate cyclic amp formation in the avian pineal gland via a novel (non-H1, non-H2, non-H3) histamine receptor subtype

The effects of histamine (HA), and selective HA, H₁-, H₂ and H₃-receptor agonists on cyclic AMP formation were investigated in intact thick and duck pineal glands. HA potently stimulated the pineal cyclic AMP formation. The effect of HA was mimicked fully by N-methylated histamines, and partially by several histaminergic drugs: 2-thiazolylethylamine (H₁), amthamine (H₂) and R-methylhistamine (H₃). Dimaprit, another selective H₂-agonist showed marginal activity. Forskolin highly potentiated the action of HA, and only weakly affected the effects of 2-thiazolyethylamine and amthamine. In the chick pineal, the stimulatory effects of HA and the tested histaminergic drugs were not blocked by mepyramine and thioperamide (H₁- and H₃-blockers, respectively), but they were antagonized by H₂-receptor selective compounds ranitidine and aminopotentidine, which, however, acted in a noncompetitive manner. Another H₂-selective blocker zolantidine antagonized the HA effect only when used at very high (30–100 μM) concentrations. In the duck pineal, the stimulatory effect of HA on cyclic AMP production was unaffected by mepyramine (H₁), thioperamide (H₃), and ranitidine (H₂), and only partially inhibited by the H₂-blocker aminopotentidine. Electrophysiological experiments revealed that HA is capable of evoking inward currents in most of the tested cells acutely isolated from chick pineal gland. The present findings further indicate that the pharmacological profile of the avian pineal HA receptor, whose stimulation leads to activation of cyclic AMP production, is different from any known HA receptor type (H₁, H₂, H₃), and suggest the existence of either an avian-specific HA receptor, or a novel HA receptor subtype. Electrophysiological data suggest that the pineal HA receptor may be somehow linked to activation of an ionic channel.     

Regulation of Norepinephrine Release from Isolated Bovine Irides by Histamine

In the present study, we investigated the effect of histamine on sympathetic neurotransmission from isolated, superfused bovine irides. We also studied the pharmacology of prejunctional histamine receptors that regulate the release of norepinephrine (NE) from this tissue. The effect of exogenous histamine and various histamine receptor agonists was examined on the release of [³H]-norepinephrine ([³H]NE) triggered by electrical field stimulation using the Superfusion Method. Histamine receptor agonists caused a concentration-dependent inhibition of field-stimulated [³H]NE overflow with the following rank order of potency: imetit > histamine > R-α-methylhistamine. In all cases, the inhibitory action of histamine receptor agonists was attenuated at high concentrations of these compounds. The histamine receptor antagonists, clobenpropit (H₃-antagonist/H₄-agonist) and thioperamide (H₃-antagonist) blocked the inhibitory response elicited by R-α-methylhistamine and imetit, respectively. Inhibitory effects of R-α-methylhistamine and clonidine were not additive suggesting that prejunctional H₃- and α₂-adrenoceptors coexist at neurotransmitter release sites. We conclude that histamine produces an inhibitory action on sympathetic neurotransmission in the bovine iris, an effect mimicked by selective H₃-receptor agonists and blocked by H₃-antagonists.     

Accelerated communication H1 contractile and H2 relaxant receptors in canine gastric muscularis mucosae

Histamine had two effects on the contractility of canine gastric muscularis mucosae in vitro: relaxation at or below 10 μM and contraction at higher concentrations. Selective agonists and antagonists were used to test the possibility that these effects were mediated by different receptor subtypes. The H₁-selective agonist 2-pyridylethylamine (2-PEA) and the H₂-selective agonist dimaprit contracted and relaxed this muscle, respectively, while the H₃-selective agonist R-α-methylhistamine had no effect. The H₁- and H₂-selective antagonists mepyramine and ranitidine selectively blocked 2-PEA-mediated contractions and dimaprit-mediated relaxations, respectively. Agonist responses, were unaltered by tetrodotoxin, suggesting a site of action other than nerves. Our results indicate that canine gastric corpus muscularis mucosae possesses both contractile H₁ and relaxant H₂ receptors.     

Role of histamine receptors in the effects of histamine on the production of reactive oxygen species by whole blood phagocytes

Aims

The diverse physiological functions of histamine are mediated through distinct histamine receptors. In this study we investigated the role of H₂R and H₄R in the effects of histamine on the production of reactive oxygen species by phagocytes in whole blood.

Main methods

Changes in reactive oxygen species (ROS) production by whole blood phagocytes after treatment with histamine, H₄R agonists (4-methylhistamine, VUF8430), H₂R agonist (dimaprit) and their combinations with H₄R antagonist (JNJ10191584) and H₂R antagonist (ranitidine) were determined using the chemiluminescence (CL) assay. To exclude the direct scavenging effects of the studied compounds on the CL response, the antioxidant properties of all compounds were measured using several methods (TRAP, ORAC, and luminol–HRP–H₂O₂ based CL).

Key findings

Histamine, 4-methylhistamine, VUF8430 and dimaprit inhibited the spontaneous and OZP-activated whole blood CL in a dose-dependent manner. On the other hand, only VUF8430 was able to inhibit PMA-activated whole blood CL. Ranitidine, but not JNJ10191584, completely reduced the effects of histamine, 4-methylhistamine and dimaprit. The direct scavenging ability of tested compounds was negligible.

Significance

Our results demonstrate that the inhibitory effects of histamine on ROS production in whole blood phagocytes were caused by H₂R. Our results also suggest that H₄R agonists in concentrations higher than 10^− 6 M may also influence ROS production via binding to H₂R.     

IDENTIFICATION OF A HISTAMINE H4 RECEPTOR ON HUMAN EOSINOPHILS—ROLE IN EOSINOPHIL CHEMOTAXIS

ABSTRACT

Eosinophils are recruited to sites of inflammation via the action of a number of chemical mediators, including PAF, leukotrienes, eotaxins, ECF-A and histamine. Although many of the cell-surface receptors for these mediators have been identified, histamine-driven chemotaxis has not been conclusively attributed to any of the three known histamine receptor subtypes, suggesting the possibility of a 4th histamine-responsive receptor on eosinophils. We have identified and cloned a novel G protein-coupled receptor (GPCR), termed Pfi-013, from an IL-5 stimulated eosinophil cDNA library which is homologous to the human histamine H₃ receptor, both at the sequence and gene structure level. Expression data indicates that Pfi-013 is predominantly expressed in peripheral blood leukocytes, with lower expression levels in spleen, testis and colon. Ligand-binding studies using Pfi-013 expressed in HEK-293Gα15 cells, demonstrates specific binding to histamine with a K_d of 3.28 ± 0.76?nM and possesses a unique rank order of potency against known histaminergic compounds in a competitive ligand-binding assay (histamine < clobenpropit < iodophenpropit < thioperamide < R-α-methylhistamine < cimetidine < pyrilamine). We have therefore termed this receptor human histamine H₄. Chemotaxis studies on isolated human eosinophils have confirmed that histamine is chemotactic and that agonists of the known histamine receptors (H₁, H₂, and H₃) do not induce such a response. Furthermore, studies employing histamine-receptor antagonists have shown an inhibition of chemotaxis only by the H₃ antagonists clobenpropit and thioperamide. Since these compounds are also antagonists of hH4 we postulate that the receptor mediating histaminergic chemotaxis is this novel histamine H₄ receptor.     

Histamine H(2) -like receptors in chick cerebral cortex: effects on cyclic AMP synthesis and characterization by [(3) H]tiotidine binding

In this study, histamine (HA) receptors in chick cerebral cortex were characterized using two approaches: (1) analysis of the effects of HA-ergic drugs on the cAMP-generating system, and (2) radioreceptor binding of [(3) H]tiotidine, a selective H(2) antagonist. HA was a weak activator of adenylyl cyclase in a crude membrane preparation of chick cerebrum. On the other hand, HA (0.1-1000 microm) potently and concentration dependently stimulated cAMP production in [(3) H]adenine pre-labelled slices of chick cerebral cortex, displaying an EC(50) value (concentration that produces 50% of maximum response) of 2.65 microm. The effect of HA was mimicked by agonists of HA receptors with the following rank order of potency: HA >or= 4-methylHA (H(2)) >or= N alpha,N alpha-dimethylHA (H(3) > H(2) = H(1)) > 2-methylHA (H(1)) > 2-thiazolylethylamine (H(1)) >or= R alpha-methylHA (H(3)) > amthamine, dimaprit (H(2)), immepip (H(3), H(4)). The HA-evoked increase in cAMP production in chick cerebral cortex was antagonized by selective H(2) receptor blockers (aminopotentidine >or= tiotidine > ranitidine > zolantidine), and not significantly affected by mepyramine and thioperamide, selective H(1) and H(3) /H(4) receptor blockers, respectively. A detailed analysis of the antagonistic action of aminopotentidine (vs. HA) revealed a non-competitive mode of action. The binding of [(3) H]tiotidine to chick cortical membranes was rapid, stable and reversible. Saturation analysis resulted in a linear Scatchard plot, suggesting binding to a single class of receptor binding site with high affinity [equilibrium dissociation constant (K (d)) = 4.42 nm] and high capacity [maximum number of binding sites (B (max) ) = 362 fmol/mg protein]. The relative rank order of HA-ergic drugs to inhibit [(3) H]tiotidine binding to chick cerebrum was: antagonists - tiotidine > aminopotentidine = ranitidine >or= zolantadine > thioperamide - triprolidine; agonists - HA >or= 4-methylHA > 2-methylHA >or=R alpha-methylHA - dimaprit. In conclusion, chick cerebral cortex contains H(2) -like HA receptors that are linked to the cAMP-generating system and are labelled with [(3) H]tiotidine. The pharmacological profile of these receptors is different from that described for their mammalian counterpart. It is suggested that the studied receptors represent either an avian-specific H(2) -like HA receptors or a novel subtype of HA receptors.     

Guanosine 5′-(γ-[35S]Thio)triphosphate Autoradiography Allows Selective Detection of Histamine H3 Receptor-Dependent G Protein Activation in Rat Brain Tissue Sections

Abstract: Histamine elicits its biological effects via three distinct G protein-coupled receptors, termed H₁, H₂, and H₃. We have used guanosine 5′-(γ-[³⁵S]thio)triphosphate (GTPγ[³⁵S]) autoradiography to localize histamine receptor-dependent G protein activation in rat brain tissue sections. Initial studies revealed that in basal conditions, adenosine was present in tissue sections in sufficient concentrations to generate an adenosine A₁ receptor-dependent GTPγ[³⁵S] signal in several brain regions. All further incubations therefore contained 8-cyclopentyl-1,3-dipropylxanthine (10 µM), a selective A₁ receptor antagonist. Histamine elicited dose-dependent increments in GTPγ[³⁵S] binding to discrete anatomical structures, most notably the caudate putamen, cerebral cortex, and substantia nigra. The overall anatomical pattern of the histamine-evoked binding response closely reflects the known distribution of H₃ binding sites and was faithfully mimicked by N^α-methylhistamine, (R)-α-methylhistamine, and immepip, three H₃-selective agonists. In all regions examined, the GTPγ[³⁵S] signal was reversed with thioperamide and clobenpropit, two potent H₃-selective antagonists, whereas mepyramine, a specific H₁ antagonist, and cimetidine, a prototypic H₂ antagonist, proved ineffective. These data indicate that in rat brain tissue sections, GTPγ[³⁵S] autoradiography selectively detects H₃ receptor-dependent signaling in response to histamine stimulation. As the existing evidence suggests that GTPγ[³⁵S] autoradiography preferentially reveals responses to G_i/o-coupled receptors, our data indicate that most, if not all, central H₃ binding sites represent functional receptors coupling to G_i/o, the inhibitory class of G proteins. Besides allowing more detailed studies on H₃ receptor signaling within anatomically restricted regions of the CNS, GTPγ[³⁵S] autoradiography offers a novel approach for functional in vitro screening of H₃ ligands.     

Histamine H<Subscript>3</Subscript> Receptor Agonists Decrease Hypothalamic Histamine Levels and Increase Stereotypical Biting in Mice Challenged with Methamphetamine

The effects of the histamine H₃ receptor agonists (R)-α-methylhistamine, imetit and immepip on methamphetamine (METH)-induced stereotypical behavior were examined in mice. The administration of METH (10 mg/kg, i.p.) to male ddY mice induced behaviors including persistent locomotion and stereotypical behaviors, which were classified into four categories: stereotypical head-bobbing (1.9%), circling (1.7%), sniffing (14.3%), and biting (82.1%). Pretreatment with (R)-α-methylhistamine (3 and 10 mg/kg, i.p.) significantly decreased stereotypical sniffing, but increased stereotypical biting induced by METH, in a dose-dependent manner. This effect of (R)-α-methylhistamine on behavior was mimicked by imetit or immepip (brain-penetrating selective histamine H₃ receptor agonists; 10 mg/kg, i.p. for each drug). Hypothalamic histamine levels 1 h after METH challenge were significantly increased in mice pretreated with saline. These increases in histamine levels were significantly decreased by pretreatment with histamine H₃ receptor agonists, effects which would appear to underlie the shift from METH-induced stereotypical sniffing to biting.     

Synthesis and structural and pharmacological properties of cyclopropane-based conformationally restricted analogs of 4-methylhistamine as histamine H3/H4 receptor ligands

On the basis of the previous results on a histamine H₄ receptor agonist 4-methylhistamine and a cyclopropane-based conformationally restricted analog CEIC (3) with potent H₃/H₄ receptor antagonistic effect, 4-methylhistamine analogs 4 and 5 of CEIC were designed and synthesized. Compound 4 showed strong affinity (K_i = 38.7 nM) for the H₃ receptor, which was more potent than a well-known H₃ antagonist thioperamide. Stable tautomer and conformation of 3 and 4, which can affect the pharmacological activity, were analyzed by ab initio calculations.     

H1- and H2-histamine receptors linked to adenylate cyclase in cell-free preparations of guinea pig cerebral cortex

Homogenates of whole or selected portions of guinea pig cerebral cortex prelabeled with [$\text{2}$-³H]adenine, were used to study the role of H₁- and H₂-histamine receptors in the activation of adenylate cyclase. Both the H₁-agonist 2-methylhistamine and the H₂-agonist 4-methylhistamine stimulated adenylate cyclase in homogenates of whole cortex and to an even greater degree in homogenates of the hippocampal portion of cortex. The H₁-antagonist tripelennamine inhibited the activation of adenylate cyclase by 2-methylhistamine but was relatively inactive against 4-methylhistamine. Whole cortex was more sensitive than hippocampus to inhibition by tripelennamine. The H₂-antagonist metiamide was equipotent against activation by 2-methylhistamine and 4-methylhistamine. The antidepressant maprotiline had the characteristics of an H₁-antagonist. This is the first cell-free study to demonstrate H₁-receptor-linked adenylate cyclase in guinea pig cerebral cortex. The results provide further support for the involvement of H₁- as well as H₂-receptors in the activation of brain adenylate cyclase.     

Histamine Stimulation of Cyclic AMP Accumulation in Astrocyte-Enriched and Neuronal Primary Cultures from Rat Brain

Histamine stimulates cyclic AMP accumulation in astrocyte-enriched and neuronal primary cultures from rat brain in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. The response in the astrocyte cultures (Emax = 304 +/- 44% over basal, EC50 = 43 +/- 5 microM) was much higher than in neuronal cultures (Emax = 24 +/- 2%, EC50 = 14 +/- 7 microM). The histamine effect in astrocytes was competitively inhibited by the H2 antagonists cimetidine (Ki = 1.1 +/- 0.2 microM) and ranitidine (Ki = 46 +/- 10 nM) but was insensitive to the H1 antagonist mepyramine (1 microM). The two selective H2 agonists impromidine and dimaprit behaved as partial agonists and showed relative potencies (139 and 0.5, respectively) consistent with an interaction with H2 receptors. The more selective H1 agonist 2-thiazolylethylamine (0.01-1 mM) did not potentiate the response to impromidine (10 microM). Thus, in contrast to what is generally observed in intact cell preparations from brain, the histamine-induced cyclic AMP accumulation in astroglial cells is mediated solely by H2 receptors. The small effect shown in neuronal cultures also appears to be mediated by H2 receptors.     

Guinea Pig Histamine H1 Receptor. II. Stable Expression in Chinese Hamster Ovary Cells Reveals the Interaction with Three Major Signal Transduction Pathways

Abstract: A cDNA encoding a guinea pig histamine H₁ receptor was stably expressed in Chinese hamster ovary (CHO) cells. In one resulting clone, named CHO(H₁), the H₁ receptor was found to be coupled to several major signal transduction pathways. In each case the involvement of a G_i/G_o protein with pertussis toxin (PTX) was assessed, as well as the influence of extracellular Ca²⁺ and of protein kinase C activation by phorbol 12-myristate 13-acetate (PMA). Histamine induced, in a PTX- and PMA-insensitive manner, a biphasic increase in the intracellular Ca²⁺ level of which only the second sustained phase was dependent on the extracellular Ca²⁺ level. Histamine also caused a threefold elevation of inositol phosphate production, which was PTX-insensitive, but slightly inhibited by PMA and reduced by 75% in the absence of extracellular Ca²⁺. Histamine also caused a massive release of arachidonic acid, which occurred in a Ca²⁺- and PMA-sensitive manner, probably through the activation of a cytosolic phospholipase A₂, which partly involves coupling to a PTX-sensitive G protein. In comparison, in HeLa cells endowed with a native H₁ receptor, the histamine-induced arachidonic acid release was also Ca²⁺- and PMA-sensitive, but totally PTX-insensitive. Finally, in CHO(H₁) cells, histamine in very low concentrations potentiated the cyclic AMP accumulation induced by forskolin. This response appeared to be insensitive to PTX, extracellular Ca²⁺, and PMA. These various observations show that stimulation of a single receptor subtype, the guinea pig H₁ receptor, can trigger four major intracellular signals through coupling to several G proteins that are variously modulated by extracellular Ca²⁺ and protein kinase C activation.     

On the role of histamine receptors in regulating pigmentary responses in Oreochromis mossambicus melanophores

Purpose: The present work was carried out to reveal the involvement of histamine receptors at the neuro-melanophore junction of teleost, Oreochromis mossambicus.

Methods: The isolated scale melanophores were assayed using the mean melanophore size index and their responses were recorded in presence of various concentrations of histamine along with H₁ and H₂ receptor specific agonists and antagonist and potentiator compound 48/80.

Results: Melanophores showed high sensitivity to histamine and its specific agonists. Histamine caused a dose-dependent pigment aggregation, whereas 2-(2-Pyridyl) ethylamine (PEA), a specific H₁R agonist also caused aggregation in a similar manner. Conversely, amthamine, a specific H₂R agonist resulted in pigment dispersion. The effects were antagonized by mepyramine; specific H₁R antagonist and ranitidine a specific H₂R antagonist.

Conclusion: It is concluded that O. mossambicus melanophores have both H₁ and H₂ receptors which mediate melanophore aggregation and dispersion respectively. Compound 48/80 augmented the melanin-aggregating and dispersing effects of PEA and amthamine. It is suggested that the effect of histamine is directly mediated through H1 and H2 receptors, whereas H1Rs may be predominantly involved in the aggregatory responses.     

Effect of Hydrogen Sulfide on Cyclic AMP Production in Isolated Bovine and Porcine Neural Retinae

Hydrogen sulfide (H₂S) has been reported to exert pharmacological effects on neural and non-neural tissues from several mammalian species. In the present study, we examined the role of the intracellular messenger, cyclic AMP in retinal response to H₂S donors, sodium hydrosulfide (NaHS) and sodium sulfide (Na₂S) in cows and pigs. Isolated bovine and porcine neural retinae were incubated in oxygenated Krebs buffer solution prior to exposure to varying concentrations of NaHS, Na₂S or the diterpene activator of adenylate cyclase, forskolin. After incubation at different time intervals, tissue homogenates were prepared for cyclic AMP assay using a well established methodology. In isolated bovine and porcine retinae, the combination of both phosphodiesterase inhibitor, IBMX (2 mM) and forskolin (10 μM) produced a synergistic increase (P < 0.001) in cyclic AMP concentrations over basal levels. NaHS (10 nM–100 μM) produced a time-dependent increase in cyclic AMP concentrations over basal levels which reached a maximum at 20 min in both bovine and porcine retinae. At this time point, both NaHS and Na₂S (10 nM–100 μM) caused a significant (P < 0.05) dose-dependent increase in cyclic AMP levels in bovine and porcine retinae. For instance, NaHS (100 nM) elicited a four-fold and three-fold increase in cyclic AMP concentrations in bovine and porcine retinae respectively whilst higher concentrations of Na₂S (100 μM) produced a much lesser effect in both species. In bovine and porcine retinae, the effects caused by forskolin (10 μM) on cyclic AMP production were not potentiated by addition of low or high concentrations of both NaHS and Na₂S. We conclude that H₂S donors can increase cyclic AMP production in isolated neural retinae from cows and pigs. Bovine retina appears to be more sensitive to the stimulatory effect of H₂S donors on cyclic nucleotide production than its porcine counterpart indicating that species differences exist in the magnitude of this response. Furthermore, effects produced by forskolin on cyclic AMP formation were not additive with those elicited by H₂S donors suggesting that these agents may share a common mechanism in their action on the adenylyl cyclase pathway.     

The mechanism of [3H]noradrenaline release by histamine and its analogs from the rat vas deferens

In this study the mechanism by which histamine and H1 and H2 agonists evoked an overflow of radioactivity from rat vasa deferentia preloaded with [3H]noradrenaline was investigated. The overflow evoked by the various agonists was unaffected by the presence of such receptor antagonists as propranolol, phentolamine, cimetidine, or scopolamine. On the other hand, the overflow evoked by all agonists except dimaprit was inhibited by mepyramine and by two well-known neuronal uptake inhibitors, cocaine and desipramine. The inhibition by mepyramine has been attributed to its effect on the neuronal uptake process. Metabolic profile studies showed that 3,4-dihydroxyphenylglycol (DOPEG) was the major constituent in the evoked overflow caused by histamine, 2-methylhistamine, 4-methylhistamine, and dimaprit and that the overflow evoked by 2-pyridylethylamine and 2-thiazolylethylamine consisted predominantly of unchanged noradrenaline. Based on these findings, it is concluded that all of the agonists tested evoke noradrenaline release intraneuronally by entering the adrenergic nerve terminals. While dimaprit might enter by passively diffusing into the adrenergic nerves, other agonists seem to use the neuronal uptake process. Noradrenaline released intraneuronally is subsequently degraded by neuronal monoamine oxidase to form DOPEG. However, there are qualitative and quantitative differences in the metabolic profile of the overflow evoked by various agonists. It is suggested that these differences could arise from their additional properties, such as their effect on the neuronal uptake process and (or) their ability to act as substrate for neuronal monoamine oxidase.     

Histaminergic H2 Action Protects Hippocampal CA1 Neurons by Prolonging the Onset of the Anoxic Depolarization in Gerbils

Abstract: The central histaminergic action on ischemia-induced neuronal damage was examined by evaluating the histological outcome and the direct current (DC) potential shift in the hippocampal CA1 region in gerbils. An intracerebroventricular administration of histamine (10–100 nmol) improved the delayed ischemic damage in hippocampal CA1 pyramidal cells produced by 3 min of transient forebrain ischemia. A high dose (75 nmol) of mepyramine, an H₁ antagonist, aggravated ischemia-induced neuronal damage, but not a low dose (0.75 nmol). Administration of cimetidine (4 nmol) and ranitidine (3 nmol), H₂ antagonists, aggravated the neuronal damage. An injection of histamine (100 nmol) prolonged the onset time of the ischemia-induced sudden shift in the extracellular DC potential (anoxic depolarization; AD) to 133% of that in control animals. Administration of mepyramine (75 nmol) did not markedly change the AD, whereas injections of cimetidine (40 nmol) and ranitidine (3 nmol) reduced the onset latency to 47 and 45%, respectively. These findings suggest that the central H₂ action serves to protect neurons by delaying the onset of AD in gerbils.     

Evaluation of the antifungal and plasma membrane H+-ATPase inhibitory action of ebselen and two ebselen analogs in S. cerevisiae cultures

The plasma membrane H⁺-ATPase pump (Pma1p) has been proposed as a viable target for antifungal drugs since this high capacity proton pump plays a critical role in the intracellular regulation of pH and in nutrient uptake of yeast and other fungi. In recent years, this and other laboratories have verified that the antifungal activity of 2-phenylbenzisoselenazol-3(2H)-one, an organoselenium compound commonly referred to as ebselen (1), stems, at least in part, from its inhibitory action on the fungal Pma1p. In the present study, the antifungal efficacy of 2-(3-pyridinyl)-benzisoselenazol-3(2H)-one (2) and 2-phenylbenzisoselenazol-3(2H)-one 1-oxide (3), two ebselen analogs, was evaluated using a strain of S. cerevisiae and compared against that of 1. In addition, the study also examined the inhibitory potential of these three compounds toward the Pma1p of S. cerevisiae. Based on mean IC₅₀ values, the antifungal potency was found to decrease in the order 3?>?1?>?2. However, in terms of inhibitory action on Pma1p, the potency decreased in the order 1?>?3?>?2. The magnitude of these activities appears to be correlated with the corresponding log P values, with compound 2 being the most hydrophilic and the least active of the three.     

Regulation of UT-A1-mediated transepithelial urea flux in MDCK cells

Transepithelial [¹⁴C]urea fluxes were measured across cultured Madin-Darby canine kidney (MDCK) cells permanently transfected to express the urea transport protein UT-A1. The urea fluxes were typically increased from a basal rate of 2 to 10 and 25 nmol·cm^–2·min^–1 in the presence of vasopressin and forskolin, respectively. Flux activation consisted of a rapid-onset component of small amplitude that leveled off within 10 min and at times even decreased again, followed by a delayed, strong increase over the next 30–40 min. Forskolin activated urea transport through activation of adenylyl cyclase; dideoxyforskolin was inactive. Vasopressin activated urea transport only from the basolateral side and was blocked by OPC-31260, indicating that its action was mediated by basolateral V₂ receptors. In the presence of the phosphodiesterase inhibitor IBMX, vasopressin activated as strongly as forskolin. By itself, IBMX caused a slow increase over 50 min to 5 nmol·cm^–2·min^–1. 8-Bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP; 300 µM) activated urea flux only when added basolaterally. IBMX augmented the activation by basolateral 8-BrcAMP. Urea flux activation by vasopressin and forskolin were only partially blocked by the protein kinase A inhibitor H-89. Even at concentrations >10 µM, urea flux after 60 min of stimulation was reduced by <50%. The rapid-onset component appeared unaffected by the presence of H-89. These data suggest that activation of transepithelial urea transport across MDCK-UT-A1 cells by forskolin and vasopressin involves cAMP as a second messenger and that it is mediated by one or more signaling pathways separate from and in addition to protein kinase A. urea transporter; Madin-Darby canine kidney cells