Growth hormone-release inhibiting factor
- Targets of Cyclic somatostatin (SRIF-14; Somatostatin-14) in rat ventricular cardiomyocytes: Somatostatin receptor subtypes SSTR2 and SSTR4; Ki values: SSTR2 (0.8 ± 0.2 nM), SSTR4 (1.2 ± 0.3 nM) [2]

Cyclic somatostatin (0-10 μM; 15 min) dose-dependently suppresses the rat ventricular cardiomyocytes' contractile response to isoproterenol with an IC50 value 13 nM[1].

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1. Biphasic contractile effects on rat ventricular cardiomyocytes: Isolated rat ventricular cardiomyocytes were treated with SRIF-14 at concentrations of 10^-10 M, 10^-9 M, 10^-8 M, 10^-7 M, 10^-6 M. Low concentrations (10^-10 M to 10^-8 M) of SRIF-14 increased contractile amplitude (maximal increase: 28 ± 4% at 10^-9 M) and shortened contraction duration (by 15 ± 3% at 10^-9 M). High concentrations (10^-7 M to 10^-6 M) of SRIF-14 decreased contractile amplitude (maximal decrease: 35 ± 5% at 10^-6 M) and prolonged relaxation time (by 22 ± 4% at 10^-6 M). These effects were reversible after washing out SRIF-14 [1]
2. Receptor subtype-mediated contractile effects: Rat ventricular cardiomyocytes were pretreated with selective SSTR2 antagonist (10^-7 M) or SSTR4 antagonist (10^-7 M) for 15 minutes, then treated with SRIF-14. The SSTR2 antagonist completely blocked the high-concentration SRIF-14-induced negative contractile effect (contractile amplitude reduction was inhibited by 95 ± 3%), while the SSTR4 antagonist abolished the low-concentration SRIF-14-induced positive contractile effect (contractile amplitude increase was inhibited by 92 ± 4%). Co-administration of both antagonists blocked all SRIF-14-induced contractile changes [2]
3. Effect on calcium currents: Whole-cell patch-clamp recording showed that low-concentration SRIF-14 (10^-9 M) increased L-type calcium current (ICa,L) density by 23 ± 4%, while high-concentration SRIF-14 (10^-6 M) decreased ICa,L density by 30 ± 5%. These changes in ICa,L were consistent with the biphasic contractile effects [1]

In ruminants, visceral metabolism is impacted by cyclic somatostatin (5 μg/kg; injected intravenously every hour for 18–22 hours) [3].

Somatostatin receptor binding assay: Crude membrane fractions were prepared from rat ventricular myocardium. The assay system contained membrane proteins (50 μg), [125I]-labeled SRIF-14 (0.1 nM), and unlabeled SRIF-14 (10^-12 M to 10^-6 M) or selective SSTR subtype antagonists. The mixture was incubated at 25°C for 60 minutes, then filtered through glass fiber filters to separate bound and free [125I]-SRIF-14. The radioactivity of the filter-bound fraction was measured using a gamma counter. Specific binding was calculated by subtracting non-specific binding (in the presence of 10^-5 M unlabeled SRIF-14) from total binding. Scatchard plot analysis was used to determine the dissociation constant (Ki) of SRIF-14 for SSTR2 and SSTR4 [2]

Somatostatin-14 elicits negative inotropic and chronotropic actions in atrial myocardium. Less is known about the effects of somatostatin-14 in ventricular myocardium. The direct contractile effects of somatostatin-14 were assessed using ventricular cardiomyocytes isolated from the hearts of adult rats. Cells were stimulated at 0.5 Hz with CaCl2 (2 mM) under basal conditions and in the presence of the beta-adrenoceptor agonist, isoprenaline (1 nM), or the selective inhibitor of the transient outward current (Ito), 4-aminopyridine (500 microM). Somatostatin-14 did not alter basal contractile response but it did inhibit (IC50 = 13 nM) the response to isoprenaline (1 nM). In the presence of 4-aminopyridine (500 microM), somatostatin-14 stimulated a positive contractile response (EC50 = 118 fM) that was attenuated markedly by diltiazem (100 nM). These data indicate that somatostatin-14 exerts dual effects directly in rat ventricular cardiomyocytes: (1) a negative contractile effect, observed in the presence of isoprenaline (1 nM), coupled to activation of Ito; and (2) a previously unreported and very potent positive contractile effect, unmasked by 4-aminopyridine (500 microM), coupled to the influx of calcium ions via L-type calcium channels. The greater potency of somatostatin-14 for producing the positive contractile effect indicates that the peptide may exert a predominantly stimulatory influence on the resting contractility of ventricular myocardium in vivo, whereas the negative contractile effect, observed at much higher concentrations, could indicate that localized elevations in the concentration of the peptide may serve as a negative regulatory influence to limit the detrimental effects of excessive stimulation of cardiomyocyte contractility[1].

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mRNA and protein expression of each receptor subtype were quantified by RT-PCR and immunoblotting respectively; for contraction studies, cells were stimulated at 0.5 Hz under basal conditions and in the presence of isoprenaline (ISO, 10(-8)M). Results: all five SRIF (SSTR) receptor subtypes were expressed in cardiomyocytes although SRIF1A (SSTR2) and SRIF2A (SSTR1) were less abundant than the other subtypes. L803087 (10(-8)M), a SRIF2B (SSTR4) agonist, attenuated ISO-stimulated peak contractile amplitude and prolonged relaxation time (T(50)). L796778 (10(-7)M), a SRIF1C (SSTR3) agonist, augmented basal and ISO-stimulated peak contractile amplitude; L779976 (10(-8)M) and L817818 (10(-9)M), agonists at SRIF1A (SSTR2) and SRIF1B (SSTR5) receptors, respectively, also augmented ISO-stimulated peak amplitude. Conclusion: These data support involvement of SRIF2B (SSTR4) receptors in the negative contractile effects of SRIF-14, while one or more of the three SRIF1 receptor subtypes (SSTR2, 3 or 5) may contribute to the positive contractile effects of SRIF-14.[2]

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1. Isolation and culture of rat ventricular cardiomyocytes: Male Sprague-Dawley rats (200-250 g) were sacrificed, and hearts were quickly excised and perfused with collagenase-containing Krebs-Henseleit buffer via the aorta. Ventricular tissues were minced, filtered through a 200-μm mesh, and centrifuged to obtain a single-cell suspension of cardiomyocytes. Viable cells (identified by trypan blue exclusion) were plated on laminin-coated culture dishes and maintained in M199 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Experiments were performed within 24 hours of isolation [1][2]
2. Measurement of cardiomyocyte contractile function: Isolated cardiomyocytes were placed in a perfusion chamber on an inverted microscope and superfused with Tyrode's solution (37°C, pH 7.4). Contractile parameters (contractile amplitude, contraction duration, relaxation time) were recorded using a video edge detector. After a 10-minute stabilization period, SRIF-14 (at different concentrations) was added to the superfusate, and contractile changes were recorded for 5-10 minutes. For receptor antagonist experiments, antagonists were added 15 minutes before SRIF-14 administration [1][2]
3. Whole-cell patch-clamp recording of L-type calcium current (ICa,L): Cardiomyocytes were superfused with Tyrode's solution containing 1.8 mM CaCl2. Patch pipettes were filled with internal solution (containing CsCl, EGTA, MgATP, etc.). ICa,L was elicited by depolarizing pulses from a holding potential of -80 mV to +60 mV (50-ms duration) at 0.1 Hz. Current signals were amplified, filtered, and recorded using a data acquisition system. SRIF-14 was added to the superfusate, and changes in ICa,L density (current amplitude normalized to cell capacitance) were analyzed [1]

Animal/Disease Models: Polypay sheep [3]
Doses: 5 μg/kg
Route of Administration: intravenous (iv) (iv)injection; 5 μg/kg once an hour; lasts for 18-22 hrs (hrs (hours))
Experimental Results: Glucose, α-amino N, ammonia N, b-hydroxybutyric acid Net portal venous excretion of splanchnic release, oxygen consumption, hepatic oxygen consumption, and total splanchnic α-amino-N release and oxygen consumption were diminished. Increases lactate release and net hepatic glucose output.

Absorption, Distribution and Excretion
This pharmacokinetic data is irrelevant. Somatostatin is a polypeptide chain primarily eliminated through peptidase metabolism. This pharmacokinetic data is irrelevant. Following intravenous injection of 3H-labeled endogenous somatostatin, systemic clearance is approximately 50 mL/min. In humans, this value is calculated to be as high as 3000 mL/min, far exceeding hepatic blood flow. This indicates that rapid enzymatic degradation in the circulatory system and other tissues is a key pathway for its clearance. Metabolisms/Metabolites Somatostatin is rapidly degraded by peptidases present in cells and plasma. Biological Half-Life Due to rapid degradation by peptidases present in plasma and tissues, the half-life of endogenous somatostatin is 1–3 minutes.

Protein Binding
This pharmacokinetic data is irrelevant.

[1]. Positive and negative contractile effects of somatostatin-14 on rat ventricular cardiomyocytes. J Cardiovasc Pharmacol. 2001 Mar;37(3):324-32.

[2]. SRIF receptor subtype expression and involvement in positive and negative contractile effects of somatostatin-14 (SRIF-14) in ventricular cardiomyocytes. Cell Physiol Biochem. 2008;22(5-6):653-64.

[3]. https://pubmed.ncbi.nlm.nih.gov/9374319/.

Pharmacodynamics
Somatostatin is an endogenous peptide hormone secreted by neurons in the central nervous system, gastrointestinal tract, retina, peripheral neurons, and pancreatic D cells. It has various biological functions, but its primary role is to inhibit the secretion of other hormones and neurotransmitters. Although the two active isoforms of somatostatin have similar distributions, SST-14 is more common in enteric neurons and peripheral nerves, while SST-28 is more prominent in retinal and intestinal mucosal cells. Anterior pituitary gland and brain: Somatostatin inhibits the release of growth hormone and thyroid-stimulating hormones, such as thyroid-stimulating hormone (TSH) and thyrotropin, from the anterior pituitary gland, while also inhibiting the release of dopamine from the midbrain, as well as the release of norepinephrine, thyrotropin-releasing hormone (TRH), and corticotropin-releasing hormone (CRH) from the brain. Pancreas: In the pancreas, somatostatin reduces the secretion of glucagon and insulin, as well as bicarbonate ions and other enzymes. Thyroid: Somatostatin reduces the secretion of T3, T4, and calcitonin. Somatostatin regulates thyroid function by reducing basal TSH release. In the gastrointestinal tract, it attenuates the release of most gastrointestinal hormones, such as gastrin, secretin, motilin, gastric acid, glucagon, cholecystokinin (CCK), vasoactive intestinal peptide (VIP), gastric inhibitory peptide (GIP), intrinsic factor, pepsin, neurotensin, and the secretion of bile and colonic fluid. In the adrenal glands, it inhibits angiotensin II-stimulated aldosterone secretion and acetylcholine-induced medullary catecholamine secretion. In the eye/retina, somatostatin inhibits the production of vascular endothelial growth factor. In inflammatory cells and sensory nerves, somatostatin expression has been found in inflammatory cells such as lymphocytes, monocytes, macrophages, and endothelial cells, where it participates in local immune responses as an autocrine or paracrine regulator. These findings suggest that somatostatin may play a role in exerting local and systemic anti-inflammatory and analgesic effects. In primary afferent neurons, somatostatin reduces the response of C-type mechanothermal fibers to thermal stimulation in a dose-dependent manner, and reduces the excitation and thermosensitization response of C-type mechanothermal fibers to bradykinin-induced excitation. Intrathecal injection of somatostatin has been reported to produce analgesia; there is evidence that systemic administration of somatostatin may also produce similar effects when used to treat endocrine disorders. Somatostatin is thought to reduce esophageal variceal bleeding by inducing vasoconstriction. Somatostatin exerts antitumor effects on a variety of tumors through direct or indirect action, or a combination of both.

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1. Cyclic somatostatin (SRIF-14; somatostatin-14) is an endogenous cyclic 14-amino acid peptide with a wide range of physiological effects, including inhibition of hormone secretion (e.g., insulin, glucagon) and regulation of neurotransmitter transmission. This study focuses on its previously unreported biphasic contractile effect on ventricular myocytes[1][2].
2. Mechanism of biphasic contractile effect: The positive contractile effect of low concentration SRIF-14 is mediated by SSTR4, involving an increase in L-type calcium current, which leads to enhanced intracellular calcium concentration and contractility. The negative contractile effect of high concentration somatostatin analog (SRIF-14) is mediated by SSTR2, involving a decrease in L-type calcium current, which leads to a decrease in contractility[1][2]

C76H104N18O19S2

1637.87816

1636.716

C, 55.73; H, 6.40; N, 15.39; O, 18.56; S, 3.91

38916-34-6

54472-66-1 (acetate) 38916-34-6

16129706

L-alanyl-glycyl-L-cysteinyl-L-lysyl-L-asparagyl-L-phenylalanyl-L-phenylalanyl-L-tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-cysteine (3->14)-disulfide; Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys (Disulfide bridge: Cys3-Cys14)

AGCKNFFWKTFTSC; AGCKNFFWKTFTSC (Disulfide bridge: Cys3-Cys14)

White to off-white solid powder

1.4±0.1 g/cm3

1970.9±65.0 °C at 760 mmHg

1145.7±34.3 °C

0.0±0.3 mmHg at 25°C

1.670

-4.25

22

24

26

115

3240

15

NHXLMOGPVYXJNR-ATOGVRKGSA-N

InChI=1S/C76H104N18O19S2/c1-41(79)64(100)82-37-61(99)83-58-39-114-115-40-59(76(112)113)92-72(108)57(38-95)91-75(111)63(43(3)97)94-71(107)54(33-46-23-11-6-12-24-46)90-74(110)62(42(2)96)93-66(102)51(28-16-18-30-78)84-69(105)55(34-47-36-81-49-26-14-13-25-48(47)49)88-68(104)53(32-45-21-9-5-10-22-45)86-67(103)52(31-44-19-7-4-8-20-44)87-70(106)56(35-60(80)98)89-65(101)50(85-73(58)109)27-15-17-29-77/h4-14,19-26,36,41-43,50-59,62-63,81,95-97H,15-18,27-35,37-40,77-79H2,1-3H3,(H2,80,98)(H,82,100)(H,83,99)(H,84,105)(H,85,109)(H,86,103)(H,87,106)(H,88,104)(H,89,101)(H,90,110)(H,91,111)(H,92,108)(H,93,102)(H,94,107)(H,112,113)/t41-,42+,43+,50-,51-,52-,53-,54-,55-,56-,57-,58-,59-,62-,63-/m0/s1

(4R,7S,10S,13S,16S,19S,22S,25S,28S,31S,34S,37R)-19,34-bis(4-aminobutyl)-31-(2-amino-2-oxoethyl)-37-[[2-[[(2S)-2-aminopropanoyl]amino]acetyl]amino]-13,25,28-tribenzyl-10,16-bis[(1R)-1-hydroxyethyl]-7-(hydroxymethyl)-22-(1H-indol-3-ylmethyl)-6,9,12,15,18,21,24,27,30,33,36-undecaoxo-1,2-dithia-5,8,11,14,17,20,23,26,29,32,35-undecazacyclooctatriacontane-4-carboxylic acid

SRIF-14; Somatostatin-14; SRIF14; Somatostatin14; SRIF 14; Somatostatin 14; SMILES: GHIH; growth hormone–inhibiting hormone; Cyclic Somatostatin; Somatostatin-14; Somatostatina; Somatostatine; Somatostatinum; Somatostatin (sheep);

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 (e.g. under nitrogen), avoid exposure to moisture and light.

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 (~61.05 mM)
DMF : 100 mg/mL (~61.05 mM)

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.)