KATP channels in cerebral hemodynamics: a systematic review of preclinical and clinical studies

Cumulative evidence suggests that ATP-sensitive potassium (KATP) channels act as a key regulator of cerebral blood flow (CBF). This implication seems to be complicated, since KATP channels are expressed in several vascular-related structures such as smooth muscle cells, endothelial cells and pericytes. In this systematic review, we searched PubMed and EMBASE for preclinical and clinical studies addressing the involvement of KATP channels in CBF regulation. A total of 216 studies were screened by title and abstract. Of these, 45 preclinical and 6 clinical studies were included. Preclinical data showed that KATP channel openers (KCOs) caused dilation of several cerebral arteries including pial arteries, the middle cerebral artery and basilar artery, and KATP channel inhibitor (KCI) glibenclamide, reversed the dilation. Glibenclamide affected neither the baseline CBF nor the baseline vascular tone. Endothelium removal from cerebral arterioles resulted in an impaired response to KCO/KCI. Clinical studies showed that KCOs dilated cerebral arteries and increased CBF, however, glibenclamide failed to attenuate these vascular changes. Endothelial KATP channels played a major role in CBF regulation. More studies investigating the role of KATP channels in CBF-related structures are needed to further elucidate their actual role in cerebral hemodynamics in humans. Systematic review registration: Prospero: CRD42023339278 (preclinical data) and CRD42022339152 (clinical data).


Introduction
Cerebral hemodynamics including cerebral blood flow (CBF) and cerebral vascular tone are vital parameters contributing to brain homeostasis (1).Dysregulation of cerebrovascular hemodynamics is involved in the pathogenesis of several neurological disorders such as stroke and migraine (2,3).The molecular mechanisms involved in the modulation of cerebral hemodynamics are complex and not entirely comprehended.
Evidence from preclinical and clinical studies implicates ATP-sensitive potassium (K ATP ) channels in the regulation of CBF and the cerebral vascular tone (4)(5)(6).K ATP channels are vastly expressed at several structures of the vasculature such as arteries, penetrating arterioles and the complex mesh of capillaries.Specifically, K ATP channels are present in smooth muscle cells (SMCs), endothelial cells (ECs) and pericytes (7-12) (Figure 1).K ATP channels link the cellular metabolic state to the plasmalemma's electrophysiology.They are 10.3389/fneur.2024.1417421Frontiers in Neurology 02 frontiersin.orgactivated during ischemia and hypoxia, causing potassium efflux, hyperpolarization and subsequently vasodilation (17)(18)(19) (Figure 2).The intricate mechanisms underpinning the involvement of K ATP channels in the regulation of cerebral hemodynamics have not been systematically reviewed.Here, we systemically review preclinical and clinical studies addressing the expression of K ATP channel in the cerebral vasculature, and their involvement in CBF regulation and cerebral vasodilation.

Methods
We searched PubMed and EMBASE for articles assessing the role of K ATP channel in the cerebral vasculature.The search was conducted on 29 January 2024, and the search string was ("K ATP channels" [MeSH Terms] OR "K ATP channel" [All Fields] OR "ATP sensitive potassium channel" [All Fields] OR "K ATP channel expression" [All Fields] OR "K ATP channel knockout" [All Fields] OR "ATP sensitive potassium channel expression" [All Fields] OR "ATP sensitive potassium channel knockout" [All Fields] AND "cerebral blood flow" [MeSH Terms] OR "cerebral blood flow" [All Fields] OR "brain blood flow" [All Fields] OR "blood flow, brain" [All Fields] OR "cerebral circulation" [All Fields] OR "cerebral circulations" [All Fields] OR "flow, brain blood" [All Fields] OR "circulation, cerebrovascular" [All Fields] OR "cerebrovascular circulation" [All Fields]).

Selection criteria and study inclusion
An a priori systematic review protocol was developed.The full protocol can be obtained from the corresponding author upon reasonable request.Two study protocols were registered in Prospero [ID-numbers: CRD42023339278 (preclinical data) and CRD42022339152 (clinical data)].We followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) reporting guidelines and the recommendations from the Cochrane Collaboration (20).The population, intervention, comparison, outcome, and study design (PICOS) approach was chosen as follows: study design, sample characteristics of the sample, intervention, comparator and outcomes.Pial artery, penetrating arteriole and capillary.The pial arterial vasculature (also known as pial collaterals or leptomeningeal anastomoses) consists of smaller arteries and arterioles that connects the three major supplying the arteries of the cerebrum: the anterior cerebral artery, the middle cerebral artery and the posterior cerebral artery (13).The pial arteries are intracranial arteries on the surface of the brain within the pia-arachnoid (leptomeninges) or glia limitans (the outmost layer of the cortex composed of glial end-feet), surrounded by cerebrospinal fluid (14) and give rise to smaller penetrating arterioles (15).An important difference in vessel architecture which might influence the CBF regulation is the number of SMC layers: penetrating arterioles contain one layer of smooth muscle while smaller pial arteries contains two to three layers of smooth muscle (16).Since K ATP channels are expressed in SMC, it is expected that these channels have a higher impact in pial arteries.To date, no studies did compare the effect of KCO/KCI between these types of vessels.CBF, cerebral blood flow; K ATP , ATP-sensitive potassium; KCI; K ATP channel inhibitor KCO; K ATP channel opener; SMC, smooth muscle cell.After removing duplicates, two investigators (HASD and LK) independently screened articles, first by title and abstract and then full text to confirm eligibility for this review.The references of the included studies were also screened.Any disagreements between the investigators were resolved through discussion.If the conflict remained, a third investigator (MMK) made the final decision.Studies were restricted to English language and both preclinical and clinical studies investigating K ATP channel opener (KCO) or K ATP channel inhibitor (KCI; Table 1) and their effects on CBF and the diameter of cerebal arteries were included.Reviews, meta-analysis, conference proceedings and case reports were excluded.For each included study, the following data were extracted: article information (title, authors, and journal), study design, characteristics of the sample intervention, technique, substances used, and outcomes.No formal meta-analysis was planned.

Results
The database search identified 294 citations of which 78 were duplicates.A total of 216 studies were screened by title and abstract and 91 were full text screened.Of these, 51 studies were included, 45 preclinical (35 studies in vivo, seven studies ex vivo, two studies in vivo and ex vivo and one study in vivo and in vitro) and six clinical studies (Figure 3).Preclinical and clinical data are summarized in Tables 2, 3, respectively.

Summary of clinical studies
KCOs have been used in clinical trials for the treatment of angina pectoris, asthma and hypertension.The most common adverse event mentioned during treatment with KCOs was headache (3,68,69).
Clinical studies assessed the effect of K ATP channels in cerebral hemodynamic in healthy participants and individuals with migraine using magnetic resonance (MR) angiography and transcranial Doppler.Intravenous infusion of KCO, levcromakalim increased CBF and dilated the MCA, the middle meningeal artery (MMA) and the superficial temporal artery (STA) (3,6,70).Glibenclamide did not affect the baseline diameter of intra-and extracerebral arteries (6).In contrast to preclinical studies, glibenclamide failed to attenuate the vasodilation induced by levcromakalim (6) or by other potent endogenous vasodilators including the calcitonin gene-related peptide (CGRP) (67,71) and the pituitary adenylate cyclase-activating polypeptide (PACAP-38) (64).

Discussion
The aim of the present study is to systematically review the involvement of K ATP channels in the cerebral vasculature and the contribution of these channels in cerebrovascular hemodynamics.The main findings are that K ATP channels are expressed in cerebral vascular SMCs, ECs and pericytes and play a key role in the regulation of CBF across species (7)(8)(9)(10)(11)(12)72).
The K ATP channel is a hetero-octameric complex consisting of four regulatory sulfonylurea receptor (SUR1, SUR2A or SUR2B) subunits and four pore-forming K + inwardly rectifying (Kir6.1 or Kir6.2) subunits (73).Different compositions of K ATP channel subunits lead to unique functions in distinct tissues (74,75) (Table 4).K ATP channels, depending on their different subunit composition, are expressed in vascular SMCs and neurons.Of note, in this systematic review, a frequently used KCO, levcromakalim, has a high affinity to the Kir6.1/SUR2B subunit in the vessels (76), while glibenclamide, a non-specific KCI, has a higher affinity to the Kir6.2/SUR1 subunit which is not present in vessels (77).

Expression of K ATP channels
K ATP channels are expressed in SMCs, ECs and pericytes.The latter are contractile cells found on the abluminal surface of the endothelial wall of capillaries (78).Two ex-vivo studies using patch-clamp electrophysiology to measure whole cell currents in brain pericytes showed that activation of K ATP channels led to hyperpolarization of pericytes, and this effect was inhibited by glibenclamide (11,58).K ATP channels expressed in the endothelium of cerebral arteries might be a key component in the regulation of CBF.Endothelium removal of cerebral arterioles significantly affected the response to K ATP channel modulators (52,53).Endothelium produces numerous vasoactive mediators, including nitric oxide (NO) that influences CBF (10).Impaired endothelial function associated with hypertension (40), diabetes mellitus (35,52), and aging (45,46) reduced the impact of KCOs/KCIs.These findings indicate that K ATP channel-induced vasodilation is endothelium-dependent.However, Janigro et al. (54) demonstrated that KCOs caused a pronounced vascular SMC-mediated and a lesser endothelium-dependent vasodilation in rats.
Inhalation of anesthetics such as isoflurane/sevoflurane or hypoxia caused dilation of cerebral pial arterioles which was inhibited by glibenclamide (32).Adenosine induced dilation of cerebral arterioles in pigs (29) and hyperpolarized retinal pericytes in mice and rats (11,58) and capillary ECs in mice (11), and administration of glibenclamide inhibited the effects of adenosine.CGRP in vivo and in vitro induced dilation of dural and pial arteries.Glibenclamide attenuated the effect of CGRP in vivo, but not in vitro (60).In healthy participants, glibenclamide had no effect on CGRP-induced headache (67).
Clinical studies demonstrated that levcromakalim dilated the MMA, the MCA and the STA in healthy humans (6) and individuals with migraine (3).In contrast to the preclinical studies, glibenclamide failed to attenuate the vascular changes induced by levcromakalim (6), PACAP-38 (64), CGRP (67) or hypercapnia (65).Of note, adenosine, CGRP and PACAP-38 are potent endogenous vasodilators which activate K ATP channels indirectly through adenylate cyclase and protein kinase A phosphorylation (80-82).One study, however, reported that hypoxia increased the anterior circulation of the brain and this   G-protein activation elicited cerebrovasodilation through interaction with K ATP channels.
All the drugs did not cause significant changes in baseline diameter.

M).
Rats (n = 28) Diameters of the basilar artery and its branches were measured through a cranial window over the ventral brain stem using a microscope equipped with a TV-camera coupled to a video monitor.

Levcromakalim and Y-26763
increased the diameter of the basilar artery and its branches which was abolished by application of glibenclamide in both adult and aged rats.
No regional heterogeneity in vasodilator response in adult rats to K ATP channel openers whereas dilator response of the large arteries due to activation of K ATP channels is impaired in aged rats.Y-26763 (10 -7.5 −10 −6 M).
The dilator responses of the branches, but not the basilar artery, were smaller in aged rats.

Immunohistochemistry for SUR1
showed minimal labeling in uninjured controls compared to 24 h after SAH in the inferomedial cortex.
Glibenclamide did not alter the cerebral hemodynamics.
(  Levcromakalim increased diameter of STA but had no significant effect on radial artery diameter or V meanMCA .
K ATP channels had no significant on V meanMCA .
Intravenous infusion of either levcromakalim or placebo (isotonic saline).
V meanMCA were measured using a transcranial Doppler.
(Continued) effect was attenuated by K ATP channel blockage with glibenclamide (66).The lack of effect of glibenclamide in clinical studies could be attributed to differences in administration routes, metabolic rate and/or tissue expression of K ATP channels across species.Basic mathematical modeling of pharmacokinetics and receptor potencies showed that the dose of glibenclamide used in clinical studies had receptor occupancy of 26% at the migraine relevant K ATP channel subtype Kir6.1/SUR2B (83).

Limitations and future perspective
The major limitations for the preclinical studies are differences in methodological approaches including subjects, designs, concentrations and formulations of different types of KCOs and KCIs, potentially affecting the reported results (Table 2).Shortcomings of clinical trials assessing the hemodynamics role of K ATP channel are (1) the use of low dose of glibenclamide, (2) including individuals from all age groups, and (3) not evaluating the long-term effect of KCOs or KCIs on cerebral hemodynamics and how endothelial dysfunction interferes with this effect.An additional question is whether K ATP channels are involved in cerebral angiogenesis.
The K ATP channel emerges to be a potential target for numerous pathological conditions such as migraine and ischemic stroke.Recent studies showed that K ATP channel activation caused headache and migraine (3), indicating that KCIs might be a novel therapeutic approach for the treatment of headache and migraine.The fact that targeting K ATP channels did not affect the baseline hemodynamic state, at least based on preclinical studies, is applicable to avoid serious adverse events.Activation of K ATP channels increased CBF after cerebral ischemia in mice (51).More experiments are needed to reveal if KCOs have a clinically meaningful effect on cerebral hypoperfusion during ischemic stroke.
Other findings with direct clinical significance are that glibenclamide attenuated peripheral arterial dilation but failed to affect cerebral hemodynamics indicating an unique biochemical difference between K ATP expressed in cerebral circulation and those expressed in peripheral arteries.
Several scenarios might underlie this difference, including expression of different SUR and Kir6 isoforms, different expression levels, post-translational modifications that render cerebral vascular K ATP channels less sensitive to KCIs and/or existence of other cerebral regulatory mechanisms with higher impact.Western blotting and quantitative PCR could be used to compare the isoforms, expression within cerebral and peripheral arteries.Patchclamp electrophysiology on isolated SMCs or ECs from the cerebral and peripheral arteries can assess the functional properties and thereby drug sensitivity.
These studies might allow a possible treatment avenue for individuals with hypertension without altering cerebral hemodynamics.Several clinical studies applied KCO to treat hypertension (68, 84-86).However, a common adverse event was headache, most likely due to changes in cephalic hemodynamics.Yet, more selective agonists are needed to avoid adverse events.The next step is the development of a selective KCO to avoid headache when treating hypertension.An agonist with high affinity to the Kir6.

Conclusion
Preclinical and clinical data from this systematic review demonstrated that K ATP channels are implicated in the regulation of cerebral hemodynamic.The main findings are that K ATP channels are expressed in cerebral vascular SMCs, ECs and pericytes.KCO increased CBF and dilated cerebral arteries in both preclinical and clinical data.Glibenclamide did not change baseline CBF and cerebral diameter in preclinical studies and did not attenuate the vasodilation induced by KCOs in clinical studies.

3
Ann et al. (22) To investigate the effect on fluid percussion brain injury (FPI) on K ATP channel activity.Cromakalim (10 −8 -10 −6 M).Pigs (n = 144) Diameters of pial arteries were measured using a video microscaler through a cranial window over the parietal cortex.Cromakalim induced dilation of pial arteries which was blunted for at least 72 h post FPI in the newborn pigs and at least 4 h post FPI in the juvenile pigs, respectively.Newborn pigs were more sensitive to traumatic vascular injury than the juvenile pigs.FPI was produced using a pendulum to strike a piston on a saline-filled cylinder.K ATP channel function was impaired to a greater extent and for a longer time period in the newborn vs. the juvenile pig. 4 Armstead et al. (23) To investigate the role of heat shock protein (HSP) in the modulation of K + channel induced pial artery dilation after FPI.Cromakalim (10 −8 M).Pigs (n = 30) Diameters of pial arteries were measured with a video microscaler through a cranial window over the parietal skull.Cromakalim and CGRP induced dilation of pial arteries.HSP-27 and HSP-70 contributed to modulation of K + channel induced pial artery dilation.CGRP (10 −6 M).Under non-FPI, coadministration of exogenous HSP-27 blunted dilation to cromakalim and CGRP.However, co-administration of exogenous HSP-70 potentiated dilation to cromakalim and CGRP.HSP-27 (1 μg/mL).FPI was produced using a pendulum to strike a piston on a saline-filled cylinder.HSP-70 (1 μg/mL).
-dependent manner.Cromakalim (10 −8 -10 −6 M).Pigs (n = 70) Diameters of pial arteries were measured with a video microscaler through a cranial window over the parietal cortex.Cromakalim dilated pial arteries, that was impaired after FPI, more in males than in females.Phenylephrine prevented impairment of K ATP channelmediated cerebrovasodilation after FPI in females.Phenylephrine (1 μg/kg/ min).FPI was produced using a pendulum to strike a piston on a saline-filled cylinder.−8 -10 −6 M).Pigs (n = 90) Diameters of pial arteries were measured using a video microscaler through a cranial window over the parietal cortex.Under non-brain injury, vasopressin co-administered with cromakalim, diminished dilation of pial arteries induced by cromakalim.Vasopressin blunted K ATP channel mediated cerebrovasodilation after FPI.Vasopressin (40 pg./mL).FPI was produced using a pendulum to strike a piston on a saline-filled cylinder.using a pendulum to strike a piston on a saline-filled cylinder.Inhaled NO prevented loss of pial artery dilation in response to cromakalim

12
Bari et  al. (31) To investigate whether cerebral vasodilation induced by aprikalim is dependent on production of NO.Aprikalim (10 −8 -10 −6 M).Piglets (n = 40) Diameters of pial arterioles were measured using a video microscaler through a cranial window over the parietal cortex.Aprikalim induced dilation of pial arterioles.However, L-NAME attenuated this dilation.Aprikalim-induced dilation of pial arterioles is mediated partly by NO.Glibenclamide (10 −5 M).N G -nitro-L-arginine methyl ester (L-NAME) (15 mg/kg).Glibenclamide did not alter baseline diameter.13 Lida et al. (32) To investigate the effects of isoflurane and sevoflurane on pial arterioles via K ATP channel activation.Isoflurane Dogs (n = 24) Diameters of pial arterioles were measured using a video micrometer through a cranial window over the parietal cortex.Inhalation or topical application of either isoflurane or sevoflurane induced dilation of pial arterioles and glibenclamide attenuated the dilation.Dilation of pial arterioles appeared to be activated by K ATP channels.Sevoflurane Glibenclamide (10 −7 -10 −5 M).Systemic (inhalation) and topical administration of isoflurane and sevoflurane.14 Wei et al. (4) To investigate the role of K + channels in the vasodilator action on pial arterioles.Pinacidil (10 −7 -10 −6 M).Cats (n = 54) Diameters of pial arterioles were measured with a Vickers image splitting device through a cranial window over the parietal cortex.Pinacidil and cromakalim dilated pial arterioles which was inhibited by glyburide.K ATP channels played a role in the vasodilation of pial arterioles.

1 -
10 μM).Rats (n = 29) Diameters of pial arterioles were measured using a video image-shearing device through a cranial window over the parietal cortex.Aprikalim produced dose-related dilation of pial arterioles in nondiabetic rats but produced constriction or/and minimal dilation of pial arterioles in diabetic rats.CBF response to hypoglycemia.CBF was determined by autoradiographic [ 14 C] iodoantipyrine (IAP) method (37).Glibenclamide blocked the increases in CBF in hypoglycemia in a dose-dependent manner.
al. (43) To investigate endotheliumderived factors on capillary ECs and K ATP channels effects on capillary flow regulation and neurovascular coupling.Pinacidil (5 mM).Mice (n = NR) In vivo 4D two-photon microscopy measured the regulation of microvascular flow in somatosensory cortex.Pinacidil induced dilation of penetrating arterioles, capillaries and precapillary sphincters.K ATP channels was found in pericytes and precapillary sphincters and had a key role for blood flow control.PNU-

Y
-26763 did not affect CBF before and after the occlusion.After 3 days, the brain was dissected into slices and infarct volume of each rat was calculated as the product of the infarct times the 2-mm thickness of each section.However, the beneficial effect of Y-26763 may be due to a direct action on neuron instead of its vasodilation effect.31 Nguyen et al. (50) To investigate the mechanisms responsible for K + dilation of resistance-size cerebral arteries.Pinacidil (10 μM).Rats (n = NR) An intact MCA was dissected from the brain and the cerebral arterioles were separated from the parenchyma.BaCl 2 and glibenclamide reduced dilations in cerebral arterioles and in the basilar artery induced by pinacidil.

K
100 μM).To test if the vasodilation was mediated by endothelial NOS activation: Vessels were pretreated with NOS inhibitors L-NNA or N G -monomethyl-

39
Li et al. (58)   To investigate the effects of adenosine on the physiology of retinal pericytes.Adenosine (5 μM).Rats (n = NR) Patch-clamp electrophysiology to monitor the whole-cells currents of intact pericytes located on micro-vessels, isolated from retinas.Hyperpolarization of retinalpericytes is due to the activation of K ATP channels by adenosine or pinacidil, an effect which was blocked by glibenclamide.

Glibenclamide ( 10
μg/kg and 0.5 μL/h) Rats (n = 35).The model of SAH involved endovascular puncture of the ICA using a 4-0 filament, produced mild-to-moderate SAH, associated with low mortality.Critical responses to SAHinflammation and an increase in barrier permeability, were significantly attenuated by block of SUR1 by glibenclamide, a selective SUR1 inhibitor.SUR1 was important in the pathophysiology of SAH.CBF were measured using Laser Doppler flowmeter affixed to the skull.Shortly after inducing SAH (<15 min), glibenclamide was administrated (loading dose of 10 μg/kg intraperitoneally and then 0.5 μL/h infusion subcutaneously).

Day 2 :Day 3 :
Oral glibenclamide followed by placebo (isotonic saline) infusion.Oral placebo (multivitamin pill) followed by placebo (isotonic saline) infusion.The participants underwent 5 MRI sessions: (time points: −20, 60, 120, 160 and 200 min).Administration of oral glibenclamide/placebo infusion at 0 min and administration of levcromakalim/ placebo infusion over 20 min at 140 min of the timeline of the study.At each MRI-session, MR angiography and phase-contrast mapping were performed.MR angiography to measure vessels: MCA, MMA and STA Glibenclamide (10 mg).

TABLE 1
An overview of KCOs and KCIs included in the studies.

TABLE 2
Summary of preclinical studies.

TABLE 3
Summary of clinical studies. Continued)