N-type Ca2+ channel

The actions of urotensin II (UII) are inhibited by the omega-conotoxin GVIA (50 nM, 50 nL) [1].

The present study aimed to reveal the effects of urotensin II (UII) on sympathetic vasomotor tone in the rostral ventrolateral medulla (RVLM). UII (0.3, 3, and 30 nmol/L, 50 nL) was microinjected into the RVLM. Blood pressure (BP), heart rate (HR), and renal sympathetic nerve activity (RSNA) were measured to determine the sympathetic vasomotor tone. BP, HR, and RSNA were simultaneously recorded after drugs had been microinjected into the RVLM. Microinjection of UII (0.3, 3, and 30 nmol/L, 50 nL) into the RVLM significantly increased BP, HR, and RSNA. Pretreatment with BIM23127 (300 nmol/L, 50 nL), a potent antagonist of the UII receptor GPR14, abolished the effect of UII. Previous microinjection of PD98059 (25 μmol/L, 50 nL), an inhibitor of ERK, significantly suppressed the effects of UII. Preinjection of an inhibitor of the N-type Ca2+ channel, ω-conotoxin GVIA (50 nmol/L, 50 nL), inhibited the effects of UII. The present study demonstrated that microinjection of UII into the RVLM significantly increased sympathetic vasomotor tone, which was mediated by the GPR14/ERK/N-type Ca2+ channel pathway. UII may become a novel therapeutic target for autonomic nervous system regulation, especially in hypertension.[1].

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The structure-function relationships of the N-type calcium channel blocker, ω-conotoxin GVIA (GVIA), have been elucidated by structural, binding and in vitro and in vivo functional studies of alanine-substituted analogues of the native molecule. Alanine was substituted at all non-bridging positions in the sequence. In most cases the structure of the analogues in aqueous solution was shown to be native-like by 1H NMR spectroscopy. Minor conformational changes observed in some cases were characterized by two-dimensional NMR. Replacement of Lys2and Tyr13 with Ala caused reductions in potency of more than 2 orders of magnitude in three functional assays (sympathetic nerve stimulation of rat isolated vas deferens, right atrium and mesenteric artery) and a rat brain membrane binding assay. Replacement of several other residues with Ala (particularly Arg17, Tyr22 and Lys24) resulted in significant reductions in potency (<100-fold) in the functional assays, but not the binding assay. The potencies of the analogues were strongly correlated between the different functional assays but not between the functional assays and the binding assay. Thus, the physiologically relevant assays employed in this study have shown that the high affinity of GVIA for the N-type calcium channel is the result of interactions between the channel binding site and the toxin at more sites than the previously identified Lys2 and Tyr13 [2].

Binding Assay [2]
Crude rat brain membranes were prepared by the method of Cruz and Olivera (29). Individual brains were homogenized using an Ultra-Turrax homogenizer (Janke and Kunkel) in 10 volumes of buffer (0.32 m sucrose, 5 mm HEPES-Tris, pH 7.4, 0.1 mm phenylmethylsulfonyl fluoride) and the homogenate spun at 1000 × g for 10 min at 4 °C. The pellet was resuspended in another 10 volumes of buffer and spun again. The combined supernatants were then spun at 17,000 × gfor 60 min at 4 °C. The pellet was taken up in 100 volumes of buffer as above but also containing 50 mg/liter lysozyme. This gives membrane derived from about 200 μg of tissue/20 μl of homogenate. The suspension was divided into 3-ml aliquots and stored at −70 °C.
Binding assays were conducted in 96-well microtiter plates with 0.65-μm filters in the bottom, using a modification of the method of Cruz and Olivera and Haack et al., as also used by Kim et al. 3 The binding buffer contained 0.32m sucrose, 5 mm HEPES-NaOH (pH 7.4), 0.1 mm phenylmethylsulfonyl fluoride, 0.3% bovine serum albumin, and 50 mg/liter lysozyme. Wash buffer consisted of 150 mm NaCl, 5 mm HEPES-NaOH, pH 7.4, 1.5 mm CaCl2, and 0.1% bovine serum albumin. Tracer solution was prepared as follows; 50 μCi of125I-[Tyr22]GVIA (DuPont NEN) was dissolved in 1 ml of water and divided into 50-μl aliquots, which were stored at −15 °C. Tracer for each assay was prepared by diluting one aliquot (2.5 μCi) into 3.5 ml of binding buffer for each 96-well microtiter plate to be used in the assay. Incubation mixtures (total volume 100 μl/well) consisted of 50 μl of binding buffer, containing displacing ligand, 20 μl of membrane preparation, and 30 μl of diluted 125I-[Tyr22]GVIA tracer solution, final concentration 0.13 nm. Nonspecific binding was determined in the presence of 1 μm unlabeled GVIA. Assays were incubated at 4 °C for 90 min and were terminated by filtration under vacuum. Each well was washed three times with 200 μl of wash buffer and then left under vacuum for 10 min to dry the filters. Filters were punched out and counted in a γ counter. Each measurement was determined in quadruplicate within an experiment, and each experiment was replicated at least three times.

Vas Deferens[2]
Vasa deferentia were mounted in 5-ml organ baths, with the top of each tissue attached to an isometric force transducer (Grass FT03) and the bottom attached to a movable support and straddled with platinum stimulating electrodes. The vasa were stretched by a passive force of about 10 millinewtons and stimulated with single electrical field pulses (100 V, 0.2-ms duration) every 20 s. The resulting twitch responses were mediated by sympathetic nerves, being sensitive to inhibition by guanethidine (10 μm) or tetrodotoxin (0.1 μm), and were recorded on a chart recorder. GVIA (0.3–10 nm) caused a gradual concentration-related reduction in the size of the twitch response to electrical nerve stimulation in the rat vas deferens. Submaximally effective concentrations of GVIA elicited an initially rapid fall in the size of the twitch, and a continued very gradual decrease thereafter for over 90 min. This apparently slow equilibration meant that concentration-response curves with full equilibration of the toxin could not be constructed without interference from spontaneous fade of the twitch responses. To offset this problem, a protocol with fixed 20-min intervals between successive concentration increments was used. Cumulative concentration-response curves for GVIA or the analogues were constructed by addition of the peptides to the solution bathing the vas deferens in 10-fold concentration increments from 1 nm. A single concentration-response curve was constructed in each tissue.

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Mesenteric Artery[2]
Under a dissecting microscope, an artery (three branch orders proximal to the arteries that enter the intestine) was carefully dissected free of the fat and connective tissue around it, and a 2-mm-long segment was mounted on 40-μm wires in a Mulvany-Halpern xstyle isometric myograph and warmed to 37 °C. The artery was incrementally stretched radially with about four steps, and the force measured and the arterial circumference calculated at each step, producing a diameter-force curve where the diameter is that of a circle with the same circumference as the vessel at each level of stretch. The diameter of the artery was then set to be 90% of the diameter predicted for distending pressure of 100 mm Hg using standard calculations. The rat mesenteric artery set up under these conditions does not develop any spontaneous active contractile force. Potassium depolarizing solution was applied for about 2 min to maximally activate the artery, and washed out. The prejunctional α2-adrenoreceptors were blocked with the covalent antagonist benextramine (3 μm for 5 min) in the presence of prazosin (0.1 μm) to protect the postjunctional α1-adrenoreceptors. The antagonists were then washed out, and noradrenaline (10 μm) was applied to confirm the washout of the antagonists. This procedure greatly increases the size of the responses to sympathetic nerve stimulation. Each jaw of the myograph was fitted with a platinum electrode about 1 mm away from the artery. Sympathetic nerves in the wall of the artery were stimulated with monopolar electrical field pulses of 0.25-ms duration at 30 V, stimulation parameters that give responses that are blocked >95% by tetrodotoxin. We have previously demonstrated that the responses are abolished by guanethidine and are thus mediated by sympathetic nerves. Stimulation of the sympathetic nerves produced contractile responses that had a short (<1 s) latency and decayed rapidly after the end of the train of stimuli. All responses were measured as the peak change in force. Nerve stimulation was applied as sets of three trains of 75 pulses at 25 Hz with a 60-s interval between trains, and 30 min between successive sets of stimuli. GVIA or analogues were applied in a cumulative fashion with 30 min of contact before each test stimulation.

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Right Atrium[2]
The right atrium was dissected free of the heart and placed in a 5-ml organ bath at 37 °C on a support having two fine platinum electrodes in contact with the atrium to collect the surface electrogram and another pair of electrodes for electrical stimulation. The spontaneous rate of contraction was continuously measured using the surface electrogram to trigger a period meter. Tachycardia responses mediated by sympathetic nerves were measured in response to sets of 4 electrical field pulses at 2 Hz, in the presence of atropine (1 μm) to abolish the effect of parasympathetic nerve stimulation. Increasing concentrations of GVIA or analogue were applied immediately following the second of two control stimulations, with the drug in contact with the tissue for 30 min before each stimulation.

Male Harlan Sprague Dawley rats were killed by CO2 anesthesia followed by decapitation, and the vasa deferentia, heart, and a portion of intestine with attached mesentery removed and placed in a dish of cool Krebs solution (composition in mm: Na+ 144, K+5.9, Mg2+ 1.2, Ca2+ 2.5, HPO4− 1.2, Cl− 129, SO4− 1.2, HCO3− 25, glucose 11, EDTA 0.026, bubbled with 5% CO2 in oxygen) for further dissection [2].

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Conscious Rabbit Experiments. The central ear artery and marginal ear vein of New Zealand White rabbits of either sex (weight 2.5 ± 0.1 kg) were cannulated under local anesthesia (1% lignocaine hydrochloride) for measurement of blood pressure (MAP) and for drug injections, respectively. The phasic blood pressure signal triggered a rate meter for the measurement of heart rate (HR). Phasic and mean arterial pressure and HR were recorded on a Grass polygraph. Following the minor procedures as outlined above, rabbits rested quietly for about 40 min in polycarbonate restrainers. The effects of intravenous administration of selected GVIA analogues were assessed on MAP, HR, and the baroreflex. The baroreflex was measured by eliciting alternate graded steady-state increases and decreases in MAP (± 5–35 mm Hg from base line) with phenylephrine and sodium nitroprusside, respectively. GVIA analogue potencies were assessed by comparing their effects to the effect of 10 μg/kg GVIA. The GVIA analogues were administered at an initial dose of 3 or 10 μg/kg. MAP and HR were monitored for 60 min and the dose of analogue increased (cumulative half-log10 dose increments) if its effect was less than that of 10 μg/kg intravenous GVIA. The baroreflex curve was then reassessed 60 min after the highest dose of peptide was administered and the reflex parameters (gain, location, and plateaus) obtained by fitting the baroreceptor-heart rate reflex curve to a logistic equation. [2]

[1]. Ya-Kun Cao, et al. Microinjection of urotensin II into the rostral ventrolateral medulla increases sympathetic vasomotor tone via the GPR14/ERK pathway in rats. Hypertens Res. 2020 Aug;43(8):765-771.
[2]. Structure-function relationships of omega-conotoxin GVIA. Synthesis, structure, calcium channel binding, and functional assay of alanine-substituted analogues. J Biol Chem. 1997 May 2;272(18):12014-23. doi: 10.1074/jbc.272.18.12014.

C120H182N38O43S6

3037.34787999999

3035.15

106375-28-4

106375-28-4

Cys-Lys-Ser-{Hyp}-Gly-Ser-Ser-Cys-Ser-{Hyp}-Thr-Ser-Tyr-Asn-Cys-Cys-Arg-Ser-Cys-Asn-{Hyp}-Tyr-Thr-Lys-Arg-Cys-Tyr-NH2 (Disulfide bridge: Cys1-Cys16; Cys8-Cys19; Cys15-Cys26)

CKS-{Hyp}-GSSCS-{Hyp}-TSYNCCRSCN-{Hyp}-YTKRCY-NH2 (Disulfide bridge: Cys1-Cys16; Cys8-Cys19; Cys15-Cys26)

White to off-white solid powder

-15.8

1496.380

C[C@](O)([H])[C@@](/N=C(O)/[C@](/N=C(O)/[C@]1([H])C[C@](O)([H])CN12)([H])CC3=CC=C(O)C=C3)([H])/C(O)=N/[C@@](/C(O)=N/[C@@](/C(O)=N\[C@@](/C(O)=N\[C@@](C(O)=N)([H])CC4=CC=C(O)C=C4)([H])CSSC[C@@](N=C(O)[C@](N=C(O)[C@](N=C(O)[C@](N=C(O)[C@](N=C(O)[C@]5([H])C[

DQZTPPHJRQRQQ-UHFFFAOYSA-N

InChI=1S/C120H182N38O43S6/c1-53(165)91-114(197)138-66(10-4-6-26-122)95(178)136-68(12-8-28-132-120(129)130)98(181)149-81(107(190)139-69(93(126)176)29-55-13-19-58(167)20-14-55)49-204-207-52-84-110(193)153-80-48-203-202-47-64(123)94(177)135-65(9-3-5-25-121)97(180)147-78(45-163)117(200)156-38-61(170)32-85(156)111(194)133-37-90(175)134-74(41-159)102(185)145-76(43-161)105(188)152-83(109(192)148-79(46-164)118(201)158-40-63(172)34-87(158)113(196)155-92(54(2)166)115(198)146-77(44-162)103(186)140-70(30-56-15-21-59(168)22-16-56)99(182)141-72(35-88(124)173)100(183)150-84)51-206-205-50-82(151-104(187)75(42-160)144-96(179)67(137-106(80)189)11-7-27-131-119(127)128)108(191)143-73(36-89(125)174)116(199)157-39-62(171)33-86(157)112(195)142-71(101(184)154-91)31-57-17-23-60(169)24-18-57/h13-24,53-54,61-87,91-92,159-172H,3-12,25-52,121-123H2,1-2H3,(H2,124,173)(H2,125,174)(H2,126,176)(H,133,194)(H,134,175)(H,135,177)(H,136,178)(H,137,189)(H,138,197)(H,139,190)(H,140,186)(H,141,182)(H,142,195)(H,143,191)(H,144,179)(H,145,185)(H,146,198)(H,147,180)(H,148,192)(H,149,181)(H,150,183)(H,151,187)(H,152,188)(H,153,193)(H,154,184)(H,155,196)(H4,127,128,131)(H4,129,130,132)

68-amino-36,71-bis(4-aminobutyl)-N-[1-amino-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]-22,51-bis(2-amino-2-oxoethyl)-33,60-bis(3-carbamimidamidopropyl)-8,47,78-trihydroxy-13,39-bis(1-hydroxyethyl)-4,16,57,74,86,89-hexakis(hydroxymethyl)-19,42-bis[(4-hydroxyphenyl)methyl]-2,5,11,14,17,20,23,32,35,38,41,44,50,53,56,59,62,69,72,75,81,84,87,90,97-pentacosaoxo-27,28,65,66,93,94-hexathia-3,6,12,15,18,21,24,31,34,37,40,43,49,52,55,58,61,70,73,76,82,85,88,91,96-pentacosazahexacyclo[52.37.4.225,63.06,10.045,49.076,80]heptanonacontane-30-carboxamide

ω-CgTx GVIA, ω-Conotoxin GVIA,Conus geographus

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O: ~100 mg/mL (TFA salt)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)