Naphthalene Sulfonamide Research Articles

Structural characterization of naphthalene sulfonamides and a sulfonate ester and their in vitro efficacy against Leishmania tarentolae promastigotes

Structural and biological activity analyses of two naphthalene sulfonamides and a naphthalene sulfonate ester.

Palladium-catalyzed cross-coupling reactions on a bromo-naphthalene scaffold in the search for novel human CC chemokine receptor 8 (CCR8) antagonists

The naphthalene sulfonamide scaffold is known to possess CCR8 antagonistic properties. In order to expand the structure–activity relationship study of this compound class, a variety of palladium-catalyzed cross-coupling reactions was performed on a bromo-naphthalene precursor yielding a diverse library. These compounds displayed CCR8 antagonistic properties in binding and calcium mobilization assays, with IC50 values in the 0.2 – 10 µM range. The decreased activity, when compared to the original lead compound, was rationalized by homology molecular modeling.

Discovery of 2-oxy-2-phenylacetic acid substituted naphthalene sulfonamide derivatives as potent KEAP1-NRF2 protein-protein interaction inhibitors for inflammatory conditions.

Nuclear factor erythroid 2-related factor 2 (NRF2) is a pleiotropic transcription factor which regulates the constitutive and inducible transcription of a wide array of genes and confers protection against a variety of pathologies. Directly disrupting Kelch-like ECH-associated protein 1 (KEAP1)-NRF2 protein-protein interaction (PPI) has been explored as a promising strategy to activate NRF2. We reported here the first identification of a series of 2-oxy-2-phenylacetic acid substituted naphthalene sulfonamide derivatives as potent KEAP1-NRF2 inhibitors. Our efforts led to the potent small molecule KEAP1-NRF2 inhibitor, 20c, which exhibited a Kd of 24nM to KEAP1 and an IC50 of 75nM in disrupting KEAP1-NRF2 interaction. Subsequent biological studies provided consistent evidence across mouse macrophage cell-based and invivo models that 20c induced NRF2 target gene expression and enhanced downstream antioxidant and anti-inflammatory activities. Our study not only demonstrated that small molecule KEAP1-NRF2 PPI inhibitors can be potential preventive and therapeutic agents for diseases and conditions involving oxidative stress and inflammation but also enriched the chemical diversity of the KEAP1-NRF2 inhibitors.

Sulfonamides as Selective NaV1.7 Inhibitors: Optimizing Potency and Pharmacokinetics to Enable in Vivo Target Engagement.

Human genetic evidence has identified the voltage-gated sodium channel NaV1.7 as an attractive target for the treatment of pain. We initially identified naphthalene sulfonamide 3 as a potent and selective inhibitor of NaV1.7. Optimization to reduce biliary clearance by balancing hydrophilicity and hydrophobicity (Log D) while maintaining NaV1.7 potency led to the identification of quinazoline 16 (AM-2099). Compound 16 demonstrated a favorable pharmacokinetic profile in rat and dog and demonstrated dose-dependent reduction of histamine-induced scratching bouts in a mouse behavioral model following oral dosing.

A Novel STAT3 Inhibitor Has Potent Activity in Preclinical Models of Acute Myeloid Leukemia That Incorporate the Stromal Environment

Background: Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with relapse rates approaching 40% in children. Aberrant activation of the Signal Transducer and Activator of Transcription 3 (STAT3) pathway is implicated in promoting many cancer phenotypes, including AML. Additionally, the important role of STAT3 in microenvironment-mediated chemoresistance is well established. Therefore, STAT3 is an important target for the development of new agents. Like other transcription factors, the structure of the STAT3 protein does not easily lend itself to the development of a small molecule inhibitor that is both potent and specific. Several commercially available and academic STAT3 inhibitors have been reported, but none is yet suitable for broad clinical application. We have developed a novel class of naphthalene sulfonamide small molecule STAT3 inhibitors with efficacy in AML cell lines and primary samples (e.g. C188-9 [Redell, et al, 2011, Blood 117:5701-9]). Rational optimization steps have yielded a new lead compound, MM-206, with improved stability in cellular contexts. Our aim is to evaluate the activity of MM-206 in preclinical AML models that include the bone marrow stromal environment.Inhibition of STAT3 activity: MM-206 potently inhibited the STAT3 SH2 domain-phosphopeptide interaction, with IC50 1.2 μM by surface plasmon resonance assays. Further, MM-206 attenuated phosphorylation of G-CSF-induced STAT3 as measured by intracellular flow cytometry. AML cell lines were treated with increasing doses of MM-206 for 30 minutes, followed by stimulation with G-CSF to induce STAT3 phosphorylation (pY705). The IC50 values for inhibition of phosphorylation were 1-3 µM, indicating potent inhibitory activity in cells. Similarly, MM-206 inhibited G-CSF-induced PIM1 transcription in a dose dependent manner in AML cell lines.Anti-leukemia activity in vitro: MM-206 demonstrated dose-dependent induction of apoptosis in AML cell lines and pediatric AML samples, even in the presence of bone marrow stromal cells. Cell lines and patient samples were cultured alone or co-cultured with mOrange-transduced HS5 or HS27A stromal cells, then treated with increasing doses of MM-206 for 24 hours. Cells were stained with Annexin V-FITC and apoptosis in the mOrange-negative cells was quantified by FACS. EC50 values were consistent with the doses for inhibition of phosphorylation, ranging from 1 - 10 µM. The increase in EC50s in the stroma co-cultured AML cells, compared to AML cells cultured alone, was ~3-fold, indicating only moderate environment-derived protection against this compound. In complementary experiments, luciferase-transduced AML cell lines were cultured alone or with stromal cells, treated with MM-206 for 24 hours, then AML cell viability was assessed by luminescence. Again, IC50s were in the range of 3 µM for all cell lines tested. There was no difference in potency for cells on stroma in the luminescence assay. In contrast to the anti-AML activity, EC50s were >30 µM in cell line models of acute lymphoblastic leukemia, a disease not typically associated with STAT3-dependent survival.Anti-leukemia activity in vivo: We engrafted NSG mice with luciferase-expressing MV4-11 AML cells. After 2 weeks, mice were randomized to receive MM-206 (30 mg/kg) or vehicle, ip daily Monday-Friday, for 2 or 4 weeks. MM-206 treatment delayed disease progression, as evidenced by significantly lower luminescence values (p<0.001, Fig. 1). Mice treated with MM-206 for 4 weeks (n=8) had significantly reduced blast percent in the bone marrow and significantly prolonged survival (p=0.02 at 10 weeks), compared to the control group (n=7).Summary: MM-206 is a novel naphthalene sulfonamide inhibitor of STAT3 activity, with potent anti-AML activity in preclinical models that incorporate the stromal environment, including a mouse xenograft model. Future work will evaluate the activity of this compound in combination with chemotherapy, as a strategy to overcome environment-mediated chemoresistance. [Display omitted] DisclosuresTweardy:StemMed, Ltd.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: compounds including MM-206, Research Funding.

Rhodium(II) Proximity‐Labeling Identifies a Novel Target Site on STAT3 for Inhibitors with Potent Anti‐Leukemia Activity

AbstractNearly 40 % of children with acute myeloid leukemia (AML) suffer relapse arising from chemoresistance, often involving upregulation of the oncoprotein STAT3 (signal transducer and activator of transcription 3). Herein, rhodium(II)‐catalyzed, proximity‐driven modification identifies the STAT3 coiled‐coil domain (CCD) as a novel ligand‐binding site, and we describe a new naphthalene sulfonamide inhibitor that targets the CCD, blocks STAT3 function, and halts its disease‐promoting effects in vitro, in tumor growth models, and in a leukemia mouse model, validating this new therapeutic target for resistant AML.

Rhodium(II) Proximity-Labeling Identifies a Novel Target Site on STAT3 for Inhibitors with Potent Anti-Leukemia Activity.

Nearly 40 % of children with acute myeloid leukemia (AML) suffer relapse arising from chemoresistance, often involving upregulation of the oncoprotein STAT3 (signal transducer and activator of transcription 3). Herein, rhodium(II)-catalyzed, proximity-driven modification identifies the STAT3 coiled-coil domain (CCD) as a novel ligand-binding site, and we describe a new naphthalene sulfonamide inhibitor that targets the CCD, blocks STAT3 function, and halts its disease-promoting effects in vitro, in tumor growth models, and in a leukemia mouse model, validating this new therapeutic target for resistant AML.

In pursuit of small molecule chemistry for calcium-permeable non-selective TRPC channels -- mirage or pot of gold?

The primary purpose of this review is to address the progress towards small molecule modulators of human Transient Receptor Potential Canonical proteins (TRPC1, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7). These proteins generate channels for calcium and sodium ion entry. They are relevant to many mammalian cell types including acinar gland cells, adipocytes, astrocytes, cardiac myocytes, cochlea hair cells, endothelial cells, epithelial cells, fibroblasts, hepatocytes, keratinocytes, leukocytes, mast cells, mesangial cells, neurones, osteoblasts, osteoclasts, platelets, podocytes, smooth muscle cells, skeletal muscle and tumour cells. There are broad-ranging positive roles of the channels in cell adhesion, migration, proliferation, survival and turning, vascular permeability, hypertrophy, wound-healing, hypo-adiponectinaemia, angiogenesis, neointimal hyperplasia, oedema, thrombosis, muscle endurance, lung hyper-responsiveness, glomerular filtration, gastrointestinal motility, pancreatitis, seizure, innate fear, motor coordination, saliva secretion, mast cell degranulation, cancer cell drug resistance, survival after myocardial infarction, efferocytosis, hypo-matrix metalloproteinase, vasoconstriction and vasodilatation. Known small molecule stimulators of the channels include hyperforin, genistein and rosiglitazone, but there is more progress with inhibitors, some of which have promising potency and selectivity. The inhibitors include 2-aminoethoxydiphenyl borate, 2-aminoquinolines, 2-aminothiazoles, fatty acids, isothiourea derivatives, naphthalene sulfonamides, N-phenylanthranilic acids, phenylethylimidazoles, piperazine/piperidine analogues, polyphenols, pyrazoles and steroids. A few of these agents are starting to be useful as tools for determining the physiological and pathophysiological functions of TRPC channels. We suggest that the pursuit of small molecule modulators for TRPC channels is important but that it requires substantial additional effort and investment before we can reap the rewards of highly potent and selective pharmacological modulators.

Activation of dimeric ABA receptors elicits guard cell closure, ABA-regulated gene expression, and drought tolerance

Abscisic acid (ABA) is an essential molecule in plant abiotic stress responses. It binds to soluble pyrabactin resistance1/PYR1-like/regulatory component of ABA receptor receptors and stabilizes them in a conformation that inhibits clade A type II C protein phosphatases; this leads to downstream SnRK2 kinase activation and numerous cellular outputs. We previously described the synthetic naphthalene sulfonamide ABA agonist pyrabactin, which activates seed ABA responses but fails to trigger substantial responses in vegetative tissues in Arabidopsis thaliana. Here we describe quinabactin, a sulfonamide ABA agonist that preferentially activates dimeric ABA receptors and possesses ABA-like potency in vivo. In Arabidopsis, the transcriptional responses induced by quinabactin are highly correlated with those induced by ABA treatments. Quinabactin treatments elicit guard cell closure, suppress water loss, and promote drought tolerance in adult Arabidopsis and soybean plants. The effects of quinabactin are sufficiently similar to those of ABA that it is able to rescue multiple phenotypes observed in the ABA-deficient mutant aba2. Genetic analyses show that quinabactin's effects in vegetative tissues are primarily mediated by dimeric ABA receptors. A PYL2-quinabactin-HAB1 X-ray crystal structure solved at 1.98-Å resolution shows that quinabactin forms a hydrogen bond with the receptor/PP2C "lock" hydrogen bond network, a structural feature absent in pyrabactin-receptor/PP2C complexes. Our results demonstrate that ABA receptors can be chemically controlled to enable plant protection against water stress and define the dimeric receptors as key targets for chemical modulation of vegetative ABA responses.

Synthesis and evaluation of non-basic inhibitors of urokinase-type plasminogen activator (uPA)

Recent drug discovery programs targeting urokinase plasminogen activator (uPA) have resulted in nonpeptidic inhibitors consisting of amidine or guanidine functional groups attached to aromatic or heteroaromatic scaffolds. There is a general problem of poor oral bioavailability of these charged inhibitors. In this paper, we report the synthesis and evaluation of a series of naphthamide and naphthalene sulfonamides as uPA inhibitors containing non-basic groups as substitute for amidine or guanidine groups.

QSAR analysis of caffeoyl naphthalene sulfonamide derivatives as HIV-1 integrase inhibitors

Human immunodeficiency virus type 1 (HIV-1) integrase is a potential target for anti-HIV therapy. It is an essential enzyme required for replication of the acquired immunodeficiency syndrome (AIDS) virus. Caffeoyl naphthalene sulfonamide derivatives act against HIV integrase and thus have the potential to become a part of an anti-HIV drug regimen. Although caffeoyl naphthalene sulfonamide derivatives have all the features required of good anti-HIV agents such as the presence of bis-catechol moieties, polyaromatic rings, and a central linker, they do Open image in new windownot perform well as anti-HIV agents in cell-based assays, that is, they do not stop viral replication at nontoxic concentration. We carried out a quantitative structure–activity relationship (QSAR) study of caffeoyl naphthalene sulfonamide derivatives via the software WIN CAChe 6.1 and STATISTICA to improve its activity. QSAR reveals that if partition coefficient, connectivity index, and shape index of these molecules are altered, the activity is likely to increase. On the basis of the QSAR model, we designed a new series of compounds, calculated the activities, and found that they were more potent than the existing compounds.

Chemically Modified Dansyl Probes: A Fluorescent Diagnostic for Ion and Proton Detection in Solution and in Polymers

[reaction: see text] The fluorescence emission intensity of the dansyl group is significantly diminished upon appending an ethyldimethylamino group to the N1 nitrogen substituent. Addition of acids and metal ions (i.e., Zn(2+)) to solutions of trimethylethylenediamine naphthalene sulfonamide (trinsyl) 2 produces a >25-fold increase in fluorescence intensity. Trinsyl probe 2 has been used as a diagnostic for the diffusion of protons and metal ions in a network polymer as well as an optical reporter for the glass transition temperature.

Assembly of gap junction channels: mechanism, effects of calmodulin antagonists and identification of connexin oligomerization determinants.

The assembly of connexins (Cxs) into gap junction intercellular communication channels was studied. An in vitro cell-free synthesis system showed that formation of the hexameric connexon hemichannels involved dimeric and tetrameric connexin intermediates. Cx32 contains two putative cytoplasmic calmodulin-binding sites, and their role in gap junction channel assembly was investigated. The oligomerization of Cx32 into connexons was reversibly inhibited by a calmodulin-binding synthetic peptide, and by W7, a naphthalene sulfonamide calmodulin antagonist. Removing the calmodulin-binding site located at the carboxyl tail of Cx32 limited connexon formation and resulted in an accumulation of intermediate connexin oligomers. This truncation mutant, Cx32Delta215, when transiently expressed in COS-7 cells, accumulated intracellularly and had failed to target to gap junctions. Immunoprecipitation studies suggested that a C-terminal sequence of Cx32 incorporating the calmodulin-binding site was required for the formation of hetero-oligomers of Cx26 and Cx32 but not for Cx32 homomeric association. A chimera, Cx32TM3CFTR, in which the third transmembrane and proposed channel lining sequence of Cx32 was substituted by a transmembrane sequence of the cystic fibrosis transmembrane conductance regulator, did not oligomerize in vitro and it accumulated intracellularly when expressed in COS-7 cells. The results indicate that amino-acid sequences in the third transmembrane domain and a calmodulin-binding domain in the cytoplasmic tail of Cx32 are likely candidates for regulating connexin oligomerization.

Substrate specificity and inhibitor sensitivity of Ca2+/S100-dependent twitchin kinases.

Myosin-associated giant protein kinases of the titin/witchin-like superfamily have previously been implicated in the regulation of muscle function, based on genetic and physiological studies. We find that recombinant constitutively active Caenorhabditis elegans and Aplysia twitchin kinase fragments differ in their catalytic activities and peptide-substrate specificities, as well as in their sensitivities to the naphthalene sulfonamide inhibitors 1-(5-chloronaphthalenesulfonyl)-1H-hexahydro-1,4-diazepine (ML-7) and 1-(5-iodonaphthalenesulfonyl)-1H-hexahydro-1,4-diazepine (ML-9). The constitutively active Aplysia twitchin kinase fragment has a remarkably high activity (Vmax > 100 mumol.min-1.mg-1) towards some substrate peptides. The autoinhibited forms of these twitchin kinases can be activated in a Ca(2+)-dependent manner by the dimeric form of the S100A1 protein (S100A1(2)). The twitchin kinase S100A1(2)-binding site can also bind Ca2+/calmodulin but neither kinase is activated by calmodulin. The data provide a functional basis for the ongoing crystallographic study of twitchin kinase fragments.

Characterisation of the voltagemactivated calcium current in the marine ciliate Euplotes vannus

We have isolated the early Ca current (I ca) from the whole cell current that activates upon depolarisations in the marine ciliate Euplotes vannus. The peak of I ca activated within 4.2 ms at depolarisations to 5 mV with an amplitude of 2.5 ± 0.35 nA and was reduced to 1.0 ± 0.14 nA ( n = 5) when the extracellular Ca concentration was changed from 10 to 1 mM. The voltage-dependent activation curve was steeper and shifted to more negative values when external Ca 2+ was replaced by Ba 2+. The early inward current inactivated with a double-exponential time course including a fast and a slow component, and no inactivation was recorded with Ba 2+. The time constants for the recovery from inactivation varied between 44 and 153 ms according to the depolarisation-dependent Ca influx. At the common resting potential of -25 mV, I ca was not steady-state inactivated; I ca half-inactivated at -14.5 mV, and totally inactivated at -5 mV. I ca was inhibited by 10 mM extracellular Cd 2+. The peptides ω-conotoxin-GVIA (20 μM), ω-conotoxin-MVIIC (600 nM), ω-agatoxin-IVA (60 nM) and calciseptine (900 nM) did not block I ca. The benzothiazepine-derivative diltiazem (100 μM) and the dihydropiridine nifedipine (100 μM) inhibited 51% and 33% of I ca, respectively. The naphthalene sulfonamide W7 reduced I ca with an inhibition coefficient of 33 μM.

Guanine Nucleotides Modulate Calcium Currents in a Marine Paramecium

Voltage-dependent calcium channels play a critical role in many cell functions and in many cell types ranging from protozoa to vertebrates. We have shown previously that guanine nucleotides modulate the calcium action potential and the duration of backward swimming in Paramecium, both indirect measurements of calcium channel function. To determine whether guanine nucleotides to indeed alter calcium currents, the inward calcium current (ICa) in Paramecium calkinsi was studied. First, the calcium current was characterized. The magnitude of ICa increased as the extracellular calcium concentration was increased from 0.5 to 50 mmol l-1, unlike the situation in freshwater species of Paramecium where the inward calcium current magnitude is maximal when extracellular calcium levels reach 1 mmol l-1. Inorganic compounds (NiCl2 at 10 mumol l-1 and CdCl2 at 1 mmol l-1) and organic compounds (naphthalene sulfonamides, W-7 and W-12-Br at 100 and 2 mumol l-1, respectively) reduced ICa. Regardless of the holding membrane potential (from -80 to -20 mV), the threshold activation for ICa was at -10 mV and the maximum value of ICa was reached at +20 mV, suggesting that there is only one type of calcium channel in P. calkinsi. Second, we injected GTP gamma S, GTP and GDP beta S into voltage-clamped cells while monitoring calcium and/or potassium currents. GTP gamma S increased the magnitude of ICa by 42 +/- 6% (mean +/- S.D., N = 5) and the effect was irreversible, GTP increased the magnitude of ICa by 37 +/- 4% (N = 4) in a reversible manner, and GDP beta S decreased ICa by 57 +/- 8% (N = 3) irreversibly. The outward potassium currents did not change when GTP gamma S was injected into the cells. These results support the hypothesis that injection of guanine nucleotides modulates the voltage-dependent calcium channel in P. calkinsi, presumably by activating G-protein-dependent processes.

Influence of calmodulin antagonists and calcium channel blockers on triiodothyronine uptake by rat hepatoma and myoblast cell lines

The influence of calcium-related mechanisms on cellular uptake of triiodothyronine (T 3) has not yet been defined, although it is known that T 3 can stimulate cellular entry of calcium. We therefore investigated the saturable uptake of [ 125I]-T 3 (10 −11 mol/L) from serum-free medium in vitro by hepatoma (H4) cells and skeletal myoblast (L6) cells to establish the calcium-dependency of this process. We studied the effects of the following three structurally distinct types of calmodulin antagonists in H4 cells: the naphthalene sulfonamides W7, W12, and W13, calmidazolium, and trifluoperazine. Uptake of [ 125I]-T 3 as a percentage of control values (n = 4, 10 −4 mol/L antagonist) was as follows: W7, 42.0% ± 3.3% ( P < .001); W12, 87.5% ± 4.5% (NS); W13, 79.5% ± 2.5% ( P < .05); calmidazolium (10 −6 mol/L, n = 8), 55.1% ± 2.2% ( P < .001); and trifluoperazine (10 −5 mol/L, n = 6), 65.7% ± 4.1% ( P < .001). To investigate whether the calmodulin sensitivity of uptake was mediated via transmembrane calcium flux, we also studied the effects of three structurally distinct types of organic calcium channel blockers in both H4 and L6 cells. [ 125I]-T 3 uptake as a percent of control values (10 −4 mol/L blocker, n = 4) was as follows: nifedipine, 8.6% ± 0.9% (H4) and 16.7% ± 7.2% (L6); verapamil, 24.6% ± 3.2% (H4) and 61.9% ± 4.2% (L6); diltiazem, 62.7% ± 3.6% (H4) and 36.1% ± 5.4% (L6); all P < .001. Eadie-Hofstee analysis indicated competitive inhibition of T 3 uptake for both calmidazolium and nifedipine. Because calmodulin antagonists can also inhibit protein kinase C, we used phorbol myristic acetate (10 −6 mol/L) to stimulate protein kinase C (30-minute incubation at 37°C) and to inhibit protein kinase C (24-hour incubation at 37°C). Stimulation of protein kinase C did not enhance T 3 uptake. Although inhibition of protein kinase C diminished T 3 uptake to 74.0% ± 2.7% of the control value (n = 4, P < .01), this effect was insufficient to explain the inhibitory effect of the calmodulin antagonists. These studies demonstrate that saturable cellular uptake of [ 125I]-T 3 is inhibited by calmodulin antagonists and calcium channel blockers. We conclude that T 3 uptake in liver and skeletal muscle cells may occur by calcium-related processes.

Calmodulin antagonists bind to sodium‐dependent high‐affinty binding sites for tritiated imipramine

AbstractEffects of calmodulin antagonists on the binding of tritiated imipramine and inhibition of serotonin uptake were tested in brain and platelet preparations. Naphthalene sulfonamidetype calmodulin antagonists generally inhibited specific [3H]‐Imipramine binding (as well as serotonin uptake) with affinities greater than or equal to those reported for binding to calmodulin itself. The binding inhibition did not require the presence of calcium and was not affected by ethylenediaminetetraacetic acid. Other calmodulin antagonists which do not have naphthalene sulfonamide structures did not inhibit [3H]‐Imipramine binding. These results raise the possibility that the structural similarity between imipramine and the naphthalene sulfonamide cerivatives may be sufficient to allow direct competition at the binding site for [3H]‐Imipramine. This is of great importance for calmodulin biochemistry since at these concentrations, the inhibitors were previously thought to interact virtually exclusively with calmodulin. © 1992 Wiley‐Liss, Inc.

ATP‐dependent Ca2+ transport and Ca2+‐ATPase activities in an enriched plasma membrane fraction from gypsy moth larval midgut tissue

AbstractA subcellular fraction enriched in plasma membranes was obtained from gypsy moth (Lymantria dispar) larval midgut tissue. Using [45Ca]2+ as a tracer, Ca2+ transport activity by membrane vesicles in the enriched fraction was measured and shown to be ATP‐dependent, with a very high affinity for Ca2+ (apparent Km for [Ca2+ free]Abbreviations used: [Ca2+free] = concentration of free (unbound) calcium ion;CaM = calmodulin; F = fraction; IOV = inside‐out membrane vesicles; W‐5 = N‐(6‐aminohexyl)‐1‐naphthalenesulfonamide; W‐7 = N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide. = 22 nM). Ca2+ transport was abolished upon addition of the calcium ionophore, A23187. Ca2+‐stimulated, Mg2+‐dependent ATPase activity peaked between 100 and 200 nM Ca2+free. Ca2+‐Mg2+‐ATPase activity was inhibited by vanadate, 2 phenothiazine drugs (trifluoperazine and chlorpromazine), and the naphthalene sulfonamide, W‐7; the related compound, W‐5, and ouabain had a negligible effect. These results suggest the presence of a high affinity plasma membrane Ca2+ pump in gypsy moth larval midgut cells and are discussed in light of earlier work involving calcium transport in isolated midguts of larval Hyalophora cecropia. Ionic and other conditions that characterize the midgut physiology of larval Lepidoptera (e.g., luminal pH; electrochemical gradient for Ca2+; effect of certain ions and inhibitors on Ca2+ transport) contrast significantly with those found in adult Diptera. The implications that these differences may have for calcium regulation are discussed. © 1992 Wiley‐Liss, Inc.

Calcium/Calmodulin Regulation of the Rat Prolactin Gene is Conferred by the Proximal Enhancer Region

The effects of the calcium/calmodulin signaling system on expression of the rat PRL gene were studied in rat pituitary GH3 cells using two specific naphthalene sulfonamide calmodulin (CaM) antagonist drugs, W7 and a more potent and more highly specific iodo-derivative, 5-iodo-1-C8. PRL (but not GH) mRNA accumulation was markedly inhibited by W7, which in coincubations abolished the stimulation normally seen with TRH. Transient transfection assays showed that expression of the reporter gene chloramphenicol acetyl transferase (CAT) linked to 5'-flanking sequences from the PRL gene was inhibited by the calcium-channel blocker verapamil and by the two CaM antagonists. The calcium effects showed partial promoter specificity, in that transcription of PRL-CAT constructs was markedly inhibited by verapamil, but the Rous sarcoma virus-CAT construct also showed significant inhibition, whereas the pBL-CAT2 construct was unaffected. Three hundred ninety five base pairs were sufficient to confer the full inhibitory effect of calcium channel blockade or CaM antagonist seen with longer constructs. The data indicate that CaM is important for PRL gene transcription, and that the effects of CaM are exerted on DNA sequences within the proximal 395bp of prolactin 5'-flanking DNA.